Substituted O6-benzyl-8-aza-guanines

ABSTRACT

The present invention provides AGT inactivating compounds, for example, substituted O 6 -benzyl-8-aza-guanines of the formula                    
     wherein R, for example, is pivaloyloxymethyl as well as pharmaceutical compositions comprising such compounds along with a pharmaceutically acceptable carrier. The present invention further provides a method of enhancing the chemotherapeutic treatment of tumor cells in a mammal with an antineoplastic alkylating agent which causes cytotoxic lesions at the O 6 -position of guanine, by administering to a mammal an effective amount of one of the aforesaid compounds, and administering to the mammal an effective amount of an antineoplastic alkylating agent which causes cytotoxic lesions at the O 6 -position of guanine.

This is a divisional of U.S. application Ser. No. 09/333,047, filed Jun.15, 1999, which is a divisional of U.S. application Ser. No. 08/849,223,filed Apr. 28, 1997, now U.S. Pat. No. 5,958,932, which is a nationalphase application of PCT/US95/09702, filed Jul. 31, 1995, which waspublished under PCT Article 21(2) in English, which is aContinuation-in-Part of U.S. application Ser. No. 08/283,953, filed Aug.1, 1994, now U.S. Pat. No. 5,525,606.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to substituted O⁶-benzylguanines,O⁶-benzyl-8-azaguanines, and 6(4)-benzyloxypyrimidines, pharmaceuticalcompositions comprising such compounds, and-methods of using suchcompounds. The subject compounds are particularly useful in inactivatingthe human DNA repair protein O⁶-alkylguanine-DNA alkyltransferase.

BACKGROUND OF THE INVENTION

The inactivation of the human DNA repair protein O⁶-alkylguanine-DNAalkyltransferase (AGT) by O⁶-benzylguanine leads to a dramaticenhancement in the cytotoxic response of human tumor cells and tumorxenografts to chemotherapeutic drugs whose mechanism of action involvesmodification of DNA guanine residues at the O⁶-position (Dolan et al.,Proc. Natl. Acad. Sci. U.S.A., 87, 5368-5372 (1990); Dolan et al.,Cancer Res., 51, 3367-3372 (1991); Dolan et al., Cancer Commun., 2,371-377 (1990); Mitchell et al., Cancer Res., 52, 1171-1175 (1992);Friedman et al., J. Natl. Cancer Inst., 84, 1926-1931 (1992); Felker etal., Cancer Chem. Pharmacol., 32, 471-476 (1993); Dolan et al., CancerChem. Pharmacol., 32, 221-225 (1993); Dolan et al., Biochem. Pharmacol.,46, 285-290 (1993)). The AGT inactivating activity of a large number ofO⁶-benzylguanine analogs have been compared with the aim of obtaininginformation about the types of substituent groups and the sites at whichthey could be attached to O⁶-benzylguanine without significantlylowering its AGT-inactivating activity (Moschel et al., J. Med. Chem.,35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37, 342-347 (1994)).While these studies led to the production of a variety of analogs thatwere as potent or somewhat less potent than O⁶-benzylguanine, none ofthe analogs were better than O⁶-benzylguanine.

Thus, there remains a need for additional compounds which are capable ofenhancing the chemotherapeutic treatment of tumor cells in a mammal withan antineoplastic alkylating agent which causes cytotoxic lesions at theO⁶-position of guanine. The present invention provides such compoundsand associated pharmaceutical compositions and treatment methods. Theseand other objects and advantages of the present invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides 7- and 8-substituted O⁶-benzylguaninederivatives, 7,8-disubstituted O⁶-benzylguanine derivatives,7,9-disubstituted benzylguanine derivatives, 8-aza-O⁶-benzylguaninederivatives, and 4(6)-substituted2-amino-5-nitro-6(4)-benzyloxypyrimidine and2-amino-5-nitroso-6(4)-benzyloxypyrimidine derivatives which have beenfound to be effective AGT inactivators, as well as pharmaceuticalcompositions comprising such derivatives along with a pharmaceuticallyacceptable carrier. The present invention further provides a method ofenhancing the chemotherapeutic treatment of tumor cells in a mammal withan antineoplastic alkylating agent which causes cytotoxic lesions at theO⁶-position of guanine, by administering to a mammal an effective amountof one of the aforesaid derivatives, 2,4-diamino-6-benzyloxy-s-triazine,5-substituted 2,4-diamino-6-benzyloxypyrimidines, or8-aza-O⁶-benzylguanine, and administering to the mammal an effectiveamount of an antineoplastic alkylating agent which causes cytotoxiclesions at the O⁶-position of guanine.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a compound of the formula I

wherein R₁ is a substituent selected from the group consisting of amino,hydroxy, C₁-C₄ alkylamino, C₁-C₄ dialkylamino, and C₁-C₄ acylamino(although, as explained in further detail below, other substituents canbe placed at this 2-position), R₂ is a substituent selected from thegroup consisting of hydrogen, C₁-C₄ alkyl, C₁-C₄ aminoalkyl, C₁-C₄hydroxyalkyl, C₁-C₄ alkylaminoalkyl, C₁-C₄ dialkylaminoalkyl, C₁-C₄cyanoalkyl, C₁-C₄ carbamoylalkyl, C₁-C₄ pivaloylalkyl, C₁-C₆alkylcarbonyloxy C₁-C₄ alkyl, C₁-C₄ alkoxyalkyl carbonyl alkyl, ribose,2′-deoxyribose, the conjugate acid form of a C₁-C₄ carboxyalkyl, and thecarboxylate anion of a C₁-C₄ carboxyalkyl as the sodium salt (although,as explained in further detail below, other substituents can be placedat this N⁹-position), and R₃ is a substituent selected from the groupconsisting of hydrogen, halo, C₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, thiol,C₁-C₄ alkylthio, trifluoromethylthio, C₁-C₄ thioacyl, hydroxy, C₁-C₄alkoxy, trifluoromethoxy, methanesulfonyloxy,trifluoromethanesulfonyloxy, C₁-C₄ acyloxy, amino, C₁-C₄ aminoalkyl,C₁-C₄ alkylamino, C₁-C₄ dialkylamino, trifluoromethylamino,ditrifluoromethylamino, aminomethanesulfonyl, C₁-C₄ aminoacyl,aminotrifluoromethylcarbonyl, formylamino, nitro, nitroso, C₁-C₄alkyldiazo, C₅-C₆ aryldiazo, trifluoromethyl, C₁-C₄ haloalkyl,halomethyl, C₁-C₄ cyanoalkyl, cyanomethyl, cyano, C₁-C₄alkyloxycarbonyl, C₁-C₄ alkylcarbonyl, phenyl, phenylcarbonyl, formyl,C₁-C₄ alkoxymethyl, phenoxymethyl, C₂-C₄ vinyl, C₂-C₄ ethynyl, andSO_(n)R′ wherein n is 0, 1, 2, or 3 and R′ is hydrogen, C₁-C₄ alkyl,amino, or phenyl, with the proviso that R₁ is not amino when both R₂ andR₃ are hydrogen, and with the proviso that R₁ is not amino ormethylamino when R₂ is ribose or 2′-deoxyribose and R₃ is hydrogen. Itis to be understood that the substituents are defined herein such thatthe group farthest from the point of attachment of the substituent isnamed first. By way of illustration, C₁-C₆ alkylcarbonyloxy C₁-C₄ alkylincludes pivaloyloxymethyl.

Suitable compounds of the above formula include those compounds whereinR₁ is selected from the group consisting of amino, hydroxy, C₁-C₄alkylamino, C₁-C₄ dialkylamino, and C₁-C₄ alkylcarbonylamino, R₂ isselected from the group consisting of hydrogen, C₁-C₄ alkyl, and C₁-C₆alkylcarbonyloxy C₁-C₄ alkyl, and R₃ is selected from the groupconsisting of amino, halo, C₁-C₄ alkyl, hydroxy, and trifluoromethyl.Other suitable compounds include those wherein R₁ is selected from thegroup consisting of amino, hydroxy, methylamino, dimethylamino, andacetylamino, R₂ is selected from the group consisting of hydrogen,methyl, and pivaloyloxymethyl, and R₃ is selected from the groupconsisting of amino, bromo, methyl, hydroxy, and. trifluoromethyl.Examples of suitable compounds include 8-amino-O⁶-benzylguanine,8-methyl-O⁶-benzylguanine, 8-hydroxy-O⁶-benzylguanine,8-bromo-O⁶-benzylguanine, 8-trifluoromethyl-O⁶-benzylguanine,O⁶-benzylxanthine, O⁶-benzyluric acid, N²-acetyl-O⁶-benzyl-8-oxoguanine,O⁶-benzyl-N²-methylguanine, O⁶-benzyl-N²,N²-dimethylguanine,6-benzyl-8-trifluoromethyl-9-methylguanine,O⁶-benzyl-8-bromo-9-methylguanine, andO⁶-benzyl-8-bromo-9-(pivaloyloxymethyl)guanine.

The present invention also provides a compound of the formula II

wherein R₁ is NO₂ or NO, and R₂ is a substituent selected from the groupconsisting of hydrogen, halo, C₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, thiol,C₁-C₄ alkylthio, trifluoromethylthio, C₁-C₄ thioacyl, hydroxy, C₁-C₄alkyloxy, trifluoromethoxy, methanesulfonyloxy,trifluoromethanesulfonyloxy, C₁-C₄ acyloxy, C₁-C₄ aminoalkyl, C₁-C₄alkylamino, C₁-C₄ dialkylamino, trifluoromethylamino,ditrifluoromethylamino, aminomethanesulfonyl, amino C₁-C₄ alkylcarbonyl,aminotrifluoromethylcarbonyl, formylamino, nitro, nitroso, C₁-C₄alkyldiazo, C₅-C₆ aryldiazo, trifluoromethyl, C₁-C₄ haloalkyl,cyanomethyl, C₁-C₄ cyanoalkyl, cyano, C₁-C₄ alkyloxycarbonyl, C₁-C₄alkylcarbonyl, phenyl, phenylcarbonyl, formyl, C₁-C₄ alkoxymethyl,phenoxymethyl, C₂-C₄ vinyl, C₂-C₄ ethynyl, and SO_(n)R′ wherein n is 0,1, 2, or 3 and R′ is hydrogen, C₁-C₄ alkyl, amino, or phenyl. Suitablecompounds include those compounds wherein R₁ is NO₂ and R₂ is hydrogenor a C₁-C₄ alkyl. Examples of suitable compounds include2-amino-4-benzyloxy-5-nitropyrimidine and2-amino-4-benzyloxy-6-methyl-5-nitropyrimidine.

The present invention further provides a compound of the formula III

wherein R is selected from the group consisting of C₁-C₄ alkyl, C₁-C₄alkyloxycarbonyl C₁-C₄ alkyl, carboxy C₁-C₄ alkyl, cyano C₁-C₄ alkyl,aminocarbonyl C₁-C₄ alkyl, hydroxy C₁-C₄ alkyl, and C₁-C₄ alkyloxy C₁-C₄alkyl. Suitable compounds of the above formula include those wherein Ris selected from the group consisting of C₁-C₄ alkyl and C₁-C₆alkylcarbonyloxy C₁-C₄ alkyl. Examples of suitable compounds include8-aza-O⁶-benzyl-9-methylguanine and8-aza-O⁶-benzyl-9-(pivaloyloxymrethyl) guanine.

The present invention further provides a compound of the formula IV

wherein R is selected from the group consisting of C₁-C₄ alkyl, C₁-C₆alkylcarbonyloxy C₁-C₄ alkyl, C₁-C₄ alkyloxycarbonyl C₁-C₄ alkyl,carboxy C₁-C₄ alkyl, cyano C₁-C₄ alkyl, aminocarbonyl C₁-C₄ alkyl,hydroxy C₁-C₄ alkyl, and C₁-C₄ alkyloxy C₁-C₄ alkyl. Suitable compoundsinclude those wherein said R is C₁-C₆ alkylcarbonyloxy C₁-C₄ alkyl. Anexample of a suitable compound is8-aza-O⁶-benzyl-7-(pivaloyloxymethyl)guanine.

The present invention further provides a compound of the formula V

wherein R₁ is selected from the group consisting of hydrogen, halo,C₁-C₄ alkyl, halo C₁-C₄ alkyl, C₁-C₆ alkylcarbonyloxy C₁-C₄ alkyl, C₁-C₄alkyloxycarbonyl C₁-C₄ alkyl, carboxy C₁-C₄ alkyl, cyano C₁-C₄ alkyl,aminocarbonyl C₁-C₄ alkyl, hydroxy C₁-C₄ alkyl, and C₁-C₄ alkyloxy C₁-C₄alkyl, and R₂ is selected from the group consisting of C₁-C₄ alkyl, haloC₁-C₄ alkyl, C₁-C₆ alkylcarbonyloxy C₁-C₄ alkyl, C₁-C₄ alkyloxycarbonyC₁-C₄ alkyl, carboxy C₁-C₄ alkyl, cyano C₁-C₄ alkyl, aminocarbonyl C₁-C₄alkyl, hydroxy C₁-C₄ is alkyl, and C₁-C₄ alkyloxy C₁-C₄ alkyl, with theproviso that when R₁ is hydrogen, R₂ is selected from the groupconsisting of halo C₁-C₄ alkyl, C₁-C₄ alkyloxy C₁-C₄ alkyl, C₁-C₆alkylcarbonyloxy C₁-C₄ alkyl, C₃-C₄ alkyloxycarbonyl C₁-C₄ alkyl,carboxy C₂-C₄ alkyl, cyano C₂-C₄ alkyl, aminocarbonyl C₂-C₄ alkyl, andhydroxy C₁-C₃ alkyl. Suitable compounds include those wherein R₁ ishydrogen or halo, and R₂ is C₁-C₆ alkylcarbonyloxy C₁-C₄ alkyl. Examplesof suitable compounds includeO⁶-benzyl-8-bromo-7-(pivaloyloxymethyl)guanine andO⁶-benzyl-7-(pivaloyloxymethyl)guanine.

The present invention additionally provides treatment methods, which aregenerally administered via pharmaceutical compositions comprising one ormore of the O⁶-substituted compounds of the present invention. Inparticular, the present invention provides a method of enhancing thechemotherapeutic treatment of tumor cells in a mammal with anantineoplastic alkylating agent that causes cytotoxic lesions at theO⁶-position of guanine, which method comprises administering to a mammalan effective amount of one or more of the aforedescribed presentinventive compounds of formulas I-V, and administering to the mammal aneffective amount of an antineoplastic alkylating agent that causescytotoxic lesions at the O⁶-position of guanine. The present inventionalso includes the method of enhancing the chemotherapeutic treatment oftumor cells in a mammal with an antineoplastic alkylating agent thatcauses cytotoxic lesions at the O⁶-position of guanine, which methodcomprises (i) administering to a mammal an effective amount of

(a) 8-aza-O⁶-benzylguanine

(b) a compound of the formula VI

wherein R is a substituent selected from the group consisting ofhydrogen, halo, C₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, thiol, C₁-C₄ alkylthio,trifluoromethylthio, C₁-C₄ thioacyl; hydroxy, C₁-C₄ alkoxy,trifluoromethoxy, methanesulfonyloxy, trifluoromethanesulfonyloxy, C₁-C₄acyloxy, amino, C₁-C₄ aminoalkyl, C₁-C₄ alkylamino, C₁-C₄ dialkylamino,trifluoromethylamino, ditrifluoromethylamino, aminomethanesulfonyl,amino C₁-C₄ alkylcarbonyl, aminotrifluoromethylcarbonyl, formylamino,nitro, nitroso, C₁-C₄ alkyldiazo, C₅-C₆ aryldiazo, trifluoromethyl,C₁-C₄ haloalkyl, halomethyl, cyanomethyl, C₁-C₄ cyanoalkyl, cyano, C₁-C₄alkyloxycarbonyl, C₁-C₄ alkylcarbonyl, phenyl, phenylcarbonyl, formyl,C₁-C₄ alkoxymethyl, phenoxymethyl, C₂-C₄ vinyl, C₂-C₄ ethynyl, andSO_(n)R′ wherein n is 0, 1, 2, or 3 and R′ is hydrogen, C₁-C₄ alkyl,amino, or phenyl, or

(c) 2,4-diamino-O⁶-benzyl-s-triazine, and (ii) administering to themammal an effective amount of an antineoplastic alkylating agent whichcauses cytotoxic lesions at the O⁶-position of guanine.

Several O⁶-substituted compounds were tested for their ability toinactivate the human DNA repair protein, O⁶-alkylguanine-DNAalkyltransferase (AGT, alkyltransferase). Two classes of compounds wereidentified as being significantly better than O⁶-benzylguanine (theprototype low-molecular-weight irradiator) in inactivating AGT in humanHT29 colon tumor cell extracts. These were 8-substitutedO⁶-benzylguanines bearing electron-withdrawing groups at the 8-positionand 5-substituted 2,4-diamino-6-benzyloxypyrimidines bearingelectron-withdrawing groups at the 5-position. The latter derivativeswere also more effective than O⁶-benzylguanine in inactivating AGT inintact HT29 colon tumor cells. Both types of compounds were as effectiveor more effective than O⁶-benzylguanine in enhancing cell killing by1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of colon, breast andprostate cancer cells grown in culture. Provided 8-substitutedO⁶-benzylguanine derivatives bearing electron-withdrawing substituentsat the 8-position and 5-substituted 2,4-diamino-6-benzyloxypyrimidinesbearing electron-withdrawing substituents at the 5-position do notexhibit undesirable toxicity, they should be superior toO⁶-benzylguanine as chemotherapeutic adjuvants for enhancing theeffectiveness of antitumor drugs whose mechanism of action involvesmodification of the O⁶-position of DNA guanine residues. The specificcompounds surveyed-for AGT inactivating activity are illustrated below.

Preparations of the 8-substituted O⁶-benzylguanine derivatives8-amino-O⁶-benzylguanine (1a) and O⁶-benzyl-8-methylguanine (1b) wereaccomplished by treating 2,8-diamino-6-chloropurine and2-amino-6-chloro-8-methylpurine, respectively, with sodium benzyloxidein benzyl alcohol. O⁶-Benzyl-8-oxoguanine(O⁶-benzyl-7,8-dihydro-8-oxoguanine, 1c) was prepared by reacting1,1′-carbonyldiimidazole with 2,4,5-triamino-6-benzyloxypyrimidine(Pfleiderer et al., Chem. Ber., 94, 12-18 (1961)). For convenience, thecompound is illustrated in the 8-hydroxy tautomeric form although itmost probably exists in solution in the 8-keto form with a hydrogenattached to the 7-nitrogen atom. O⁶-Benzyl-8-bromoguanine (1d) wasprepared by brominatlon of O⁶-benzylguanine.O⁶-Benzyl-8-trifluoromethylguanine (1e) was prepared by reacting2-amino-6-chloro-8-trifluoro-methylpurine with sodium benzyloxide inbenzyl alcohol. 8-Aza-O⁶-benzylguanine (2) was prepared through ridrousacid treatment of 2,4, 5-triamina-6-benzyloxypyrimidine. Compound 2 hadbeen prepared previously by another route (Shealy et al., J. Org. Chem.,27, 4518-4523 (1962)).

With respect to the pyrimidine derivatives (3a-f),4-amino-6-benzyloxy-5-nitropyrimidine (3a) was prepared by treating4-amino-6-chloro-5-nitropyrimidine (Boon et al., J. Chem. Soc., 96-102(1951)) with sodium benzyloxide in benzyl alcohol. Derivatives 3b-d wereprepared by the method of Pfleiderer et al. (Chem. Ber., 94, 12-18(1961)). 2,4-Diamino-6-benzyloxy-5-nitropyrimidine (3e) and2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f) were prepared previouslyby Kosary et al. (Acta Pharm. Hung., 49, 241-247 (1989)).

The purines, O⁶-benzylxanthine (4a) and O⁶-benzyluric acid (4b) wereprepared by nitrous acid domination of O⁶-benzylguanine andO⁶-benzyl-8-oxoguanine, respectively. N²-Acetyl-O⁶-benzyl-8-oxoguanine(N²-acetyl-O⁶-benzyl-7,8-dihydro-8-oxoguanine) (4d) was prepared throughacetylation of O⁶-benzyl-8-oxoguanine (1c).O⁶-Benzyl-2-fluorohypoxanthine (4c) was prepared previously by Robinsand Robins (J. Org. Chem., 34, 2160-2163 (1969)). This material wastreated with methylamine and dimethylamine to produceO⁶-benzyl-N²-methylguanine (4e) and O⁶-benzyl-N²,N²-dimethylguanine(4f), respectively.

Compounds 5a (2-amino-4-benzyloxy-5-nitropyrimidine) and 5b(2-amino-4-benzyloxy-6-methyl-5-nitropyrimidine) were prepared bytreating 2-amino-4-chloro-5-nitropyrimidine and2-amino-4-chloro-6-methyl-5-nitropyrimidine (Boon et al., J. Chem. Soc.,96-102 (1951)), respectively, with sodium benzyloxide in benzyl alcohol.Compound 6 (2,4-diamino-6-benzyloxy-s-triazine) was prepared previouslyunder similar conditions (Wakabayashi et al., Nippon Dojo-HiryyogakuZasshi, 41, 193-200 (1970)). O⁶-Benzyl-8-trifluoromethyl-9-methylguanine(7) was prepared by treating the anion of 1e with methyl iodide inN,N-dimethylformamide.

Compound 8a was prepared by methylating the sodium salt of8-aza-O⁶-benzylguanine using methyl iodide as the methylating agent.Compounds 8b and 9 were prepared by the reaction of the sodium salt of8-aza-O⁶-benzylguanine and chloromethyl pivalate. Compound 10a wasprepared by direct bromination of O⁶-benzyl-9-methylguanine. Compounds10b and 11 were prepared by the reaction of the sodium salt ofO⁶-benzyl-8-bromoguanine and chloromethyl pivalate. Compound 12 wasprepared by the reaction of the sodium salt of O⁶-benzylguanine andchloromethyl pivalate.

The ability of these compounds to inactivate the AGT protein in HT29human colon tumor cell extracts and in intact HT29 cells is summarizedin Tables 1 and 2. The data represent the dose of compound required toproduce 50% inactivation in cell-free extracts upon incubation for 30min or in cells upon incubation for 4 hr.

TABLE 1 AGT-Inactivating Activity of 6-Benzyloxypurine,6(4)-Benzyloxypyrimidine, and 6-Benzyloxy-s-triazine Derivatives ED₅₀(μM)^(a) In HT29 In cell-free HT29 Compound extract cells2,4-diamino-6-benzyloxy-5- 0.06 0.02 nitrosopyrimidine (3d)2,4-diamino-6-benzyloxy-5-nitropyrimidine 0.06 0.02 (3e)8-aza-O⁶-benzylguanine (2) 0.07 0.06 O⁶-benzyl-8-bromoguanine (1d) 0.080.06 O⁶-benzylguanine 0.2 0.05 O⁶-benzyl-8-methyl-guanine (1b) 0.3 0.1O⁶-benzyl-8-oxoguanine (1c) 0.3 0.15 O⁶-benzyl-8-trifluoromethylguanine(1e) 0.4 0.25 2,4,5-triamino-6-benzyloxypyrimidine (3c) 0.4 0.32-amino-4-benzyloxy-6-methyl-5- 0.4 0.06 nitropyrimidine (5b)2-amino-4-benzyloxy-5-nitropyrimidine (5a) 0.4 0.058-amino-O⁶-benzylguanine (1a) 0.7 22,4-diamino-6-benzyloxy-5-bromopyrimidine 2 0.8 (3f)2,4-diamino-6-benzyloxy-s-triazine (6) 4 1.02,4-diamino-6-benzyloxypyrimidine (3b) 15 5 O⁶-benzyluric acid (4b) 2545 4-amino-6-benzyloxy-5-nitropyrimidine (3a) 28 8O⁶-benzyl-2-fluorohypoxanthine (4c) 48 12 O⁶-benzylxanthine (4a) 60 35N²-acetyl-O⁶-benzyl-8-oxoguanine (4d) 65 11 O⁶-benzyl-N²-methylguanine(4e) 160 60 O⁶-benzyl-N²,N²-dimethylguanine (4f) 200 110 ^(a)Theeffective dose required to produce 50% inactivation in cell-freeextracts upon incubation for 30 min or in cells upon incubation for 4hr. The values for O⁶-benzylguanine are from Moschel et al., J. Med.Chem., 35, 4486-4491 (1992) .

Within these series of compounds, O⁶-benzyl-N²-methyl-andO⁶-benzyl-N²,N²-dimethylguanine (4e and 4f) were the least active agentsexhibiting so values for inactivation of AGT in HT29 cell extracts of160 and 200 mM, respectively. For comparison, the ED₅₀ value exhibitedby O⁶-benzylguanine was 0.2 mM (Table 1). The other 2- and/or8-substituted 6-benzyloxypurines, N²-acetyl-O⁶-benzyl-8-oxoguarine (4d),O⁶-benzylxanthine (4a), O⁶-benzyl-2-fluorohypoxanthine (4c) andO⁶-benzyluric acid (4b), together with the substituted pyrimidines4-amino-6-benzyloxy-5-nitropyrimidine (3a) and2,4-diamino-6-benzyloxypyrimidine (3b), comprised a group ofincreasingly more active AGT inactivating agents exhibiting intermediateED₅₀ values in the range of 65 to 15 mM.2,4-Diamino-6-benzyloxy-5-triazine (6) and2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f) were considerably moreactive than 3b indicating that electron-withdrawing groups at the5-position of a 2,4-diamino-6-benzyloxypyrimidine derivative arepositive contributors to efficient AGC inactivation. This is furtheremphasized by the very high activity exhibited by2,4-diamino-6-benzyloxy-5-nitrasopyrimidine (3d) and2,4-diamino-6-benzyloxy-5-nitropyrimidine (3e), which contain stronglyelectron-withdrawing nitroso and nitro substituents, respectively. Thesetwo derivatives are the most active AGT inactivators tested to date. Theobservation that 2-amino-4-benzyloxy-5-nitropyrimidine (5a) is much moreactive than 3a indicates that a 2-amino group is critical for highactivity for a 6(4)-benzyloxy-5-nitropyrimidine derivative. Anadditional alkyl group at the 4(6)-position (e.g., as in 5b) does notenhance activity significantly over that for 5a although an amino groupat the 4(6)-position significantly enhances activity. Thus, AGTinactivating activity increases substantially over the series5a=5b<3d=3e. With these considerations in mind the activity of2,4,5-triamino-6-benzyloxypyrimidine (3c) seems exceptional and thereasons for its relatively high activity are unclear at present. It isalso significant that pyrimidines 5a and 5b are quite active in cells,which is not totally predicted by their corresponding activity inHT29cell extracts.

All the O⁶-benzylguanine analogs 1a-d were much more active than thepurines in the series 4a-f and the activity differences among 1a-d alsoreflect enhancements due to introduction of electron withdrawing groups.Thus, activity increased in the series 8-amino-O⁶-benzylguanine (1a)<O⁶-benzyl-8-axoguanine (1c) <O⁶-benzyl-8-methylguanine (1b)<O⁶-benzyl-8-bromoguanine (1d) <8-aza-O⁶-benzylguanine (2). Indeed,derivatives 1d and 2 were essentially as active as pyrimidines 3d and 3ein cell-free extracts although 1d and 2 were somewhat less active incells than expected from their activity in cell-free extracts.

The compounds listed in Table 2 also had AGT-inactivating activity incell free extracts and in cells. The activity of 7, 8a, and 10a in cellsis significantly higher than their activity in cell-free extracts. Thusthe ratio of ED50 values in cell-free extracts/intact cells is 1.6, 1.6,1.1, respectively, for derivatives id,. 1e, and 2 (Table 1). This ratioincreases to 7.2, 6.3, and 6.3, respectively, for the correspondingmethylated derivatives 10a, 7, and 8a. It is believed that the higheractivity of the methylated derivatives in the cells is due to the factthat these compounds do not possess readily dissociable hydrogens in theimidazole portion of the purine ring system and therefore they canreadily enter the cells as neutral molecules.

TABLE 2 AGT-Inactivating Activity of 7, 8- and 8, 9- DisubstitutedO⁶-Benzylguanine Derivatives and Related Compounds ED₅₀ value (μM)^(a)In HT29 In cell-free HT29 Compound extract cellsO⁶-benzyl-8-trifluoromethyl-9-methylguanine 2.5 0.4 (7)8-aza-O⁶-benzyl-9-methylguanine (8a) 0.5 0.08 8-aza-O⁶-benzyl-9-0.28^(b) 0.23 (pivaloyloxymethyl)guanine (8b) 8-aza-O⁶-benzyl-7-0.11^(c) 0.16 (pivaloyloxymethyl)guanine (9)O⁶-benzyl-8-bromo-9-methylguanine (10a) 1.9 0.25 O⁶-benzyl-8-bromo-9-0.08^(d) 0.05 (pivaloyloxymethyl)guanine (10b)O⁶-benzyl-7-(pivaloyloxymethyl)guanine (12) 9^(e) 0.3O⁶-benzyl-9-(pivaloyloxymethyl)guanine 3.1^(f,g) 0.3O⁶-benzyl-9-methylguanine 2.6^(g) 0.4 ^(a)The dose required to produce50% inactivation in cell-free extracts upon incubation for 30 min. or incells upon incubation for 4 hr. ^(b)ED₅₀ = 4 with purified human AGT.^(c)ED₅₀ = 2 with purified human AGT. ^(d)ED₅₀ > 100 with purified humanAGT. ^(e)ED₅₀ >> 100 with purified human AGT. ^(f)ED₅₀ = 95 withpurified human AGT. ^(g)Data from Chae et al., J. Med. Chem., 37,342-347 (1994) .

The ability of increasing concentrations of 1a-d, 2, and 3c-e to enhancethe killing of human HT29 colon cancer cells, DU-145 prostate cancercells, and MCF-71 breast cancer cells by BCNU (40 μM) is shown in Tables3, 4, and 5, respectively. The data reflect the number of cell coloniesthat result following exposure to AGT inactivator alone or AGTinactivator 2 hr before exposure to BCNU as described in Dolan et al.(Proc. Natl. Acad. Sci., U.S.A., 87, 5368-5372 (1990)). Data forO⁶-benzylguanine are included for comparison. As indicated, at 10 μMconcentrations, all the 8-substituted purines with the exception of lawere as effective as O⁶-benzylguanine in enhancing the cytotoxicity ofBCNU (40 μM); such treatment killed essentially all the tumor cells.Treatment of the cells with the modified 8-substituted O⁶-benzylguaninealone or BCNU alone had no significant effect on cell colony number. Thecomparatively low activity of 1a in all but the breast cancer cells mayreflect its poor transport into other tumor cell types or its rapidmetabolic conversion to an ineffective AGT inactivator. Its ineffectiveenhancement of BCNU cytotoxicity parallels its relatively poor AGTinactivating ability in colon tumor cells (Table 1).

For the pyrimidines tested, 2,4,5-triamino-6-benzyloxypyrimidine (3c)was as effective as the 8-substituted O⁶-bezylguanine derivatives andO⁶-benzylguanine itself in enhancing BCNU toxicity although the nitroso-and nitropyrimldine derivatives (3d and 3e) were similarly effective ata 4-fold lower dose.

TABLE 3 Killing of HT-29 Colon Cancer Cells by BCNU Combined with AGTInactivators Colony Inactivator Formation Concentration BCNU per 1000Inactivator (μM) (μM) cells None None 435 ± 63 None 40 442 ± 34O⁶-benzylguanine 10 None 431 ± 33 10 40 13 ± 6 2.5 40  38 ± 15 1 40 277± 25 8-aza-O⁶-benzylguanine 10 None 537 ± 48 (2) 10 40  2 ± 1 1 40 423 ±42 O⁶-benzyl-8-bromoguanine 10 None 401 ± 22 (1d) 10 40  1 ± 0 1 40 299± 30 O⁶-benzyl-8-oxoguanine 10 None 401 ± 22 (1c) 10 40 <1 1 40 221 ± 15O⁶-benzyl-8- 10 None 513 ± 76 methylguanine (1b) 10 40 <1 1 40 230 ± 51O⁶-benzyl-8-aminoguanine 10 None 504 ± 30 (1a) 10 40 430 ± 41 1 40 475 ±26 2,4,5-triamino-6- 10 None 453 ± 59 benzyloxypyrimidine (3c) 10 40  3± 1 1 40 487 ± 32 2,4-diamino-6-benzyloxy- 2.5 None 528 ± 645-nitrosopyrimidine (3d) 2.5 40 <1 1 40 19 ± 4 2,4-diamino-6-benzyloxy-2.5 None 438 ± 25 5-nitropyrimidine (3e) 2.5 40 <1 1 40 45 ± 4

TABLE 4 Killing of DU-145 Prostate Cancer Cells by BCNU Combined withAGT Inactivators Colony Inactivator Formation Concentration BCNU per1000 Inactivator (μM) (μM) cells None None 453 ± 81 None 40 394 ± 76O⁶-benzylguanine 10 None 462 ± 68 10 40 28 ± 5 1 40 299 ± 188-aza-O⁶-benzylguanine 10 None 452 ± 72 (2) 10 40 28 ± 5 1 40 248 ± 21O⁶-benzyl-8- 10 None 493 ± 90 bromoguanine (1d) 10 40 16 ± 3 1 40 267 ±39 O⁶-benzyl-8-oxoguanine 10 None 379 ± 34 (1c) 10 40 34 ± 3 1 40 329 ±43 O⁶-benzyl-8- 10 None 357 ± 43 methylguanine (1b) 10 40 50 ± 7 1 40 306 ± 157 O⁶-benzyl-8- 10 None 380 ± 36 aminoguanine (1a) 10 40 435 ±70 1 40 295 ± 45 2,4,5-triamino-6- 10 None  429 ± 101benzyloxypyrimidine 10 40 57 ± 7 (3c) 1 40 378 ± 60 2,4-diamino-6- 2.5None 403 ± 35 benzyloxy-5- 2.5 40  7 ± 3 nitrosopyrimidine 1 40 25 ± 4(3d) 0.25 40 192 ± 17 2,4-diamino-6- 2.5 None 407 ± 80 benzyloxy-5- 2.540  9 ± 2 nitropyrimidine (3e) 1 40 59 ± 6 0.25 40 129 ± 26

TABLE 5 Killing of MCF-71 Breast Cancer Cells by BCNU Combined with AGTInactivators Colony Inactivator Formation Concentration BCNU per 1000Inactivator (μM) (μM) cells None None 426 ± 78 None 40 364 ± 60O⁶-benzylguanine 10 None 455 ± 63 10 40  4 ± 2 2.5 40 12 ± 68-aza-O⁶-benzylguanine 10 None 483 ± 27 (2) 10 40  2 ± 1O⁶-benzyl-8-bromoguanine 10 None  380 ± 109 (1d) 10 40  3 ± 1 2.5 40  4± 3 O⁶-benzyl-8-oxoguanine 10 None 522 ± 78 (1c) 10 40  4 ± 2O⁶-benzyl-8-methylguanine 10 None 376 ± 76 (1b) 10 40  2 ± 1O⁶-benzyl-8-aminoguanine 10 None 432 ± 36 (1a) 10 40 95 ± 82,4,5-triamino-6- 10 None 448 ± 55 benzyloxypyrimidine (3c) 10 40 12 ± 42,4-diamino-6-benzyloxy- 2.5 None 447 ± 87 5-nitrosopyrimidine (3d) 2.540  2 ± 1 2,4-diamino-6-benzyloxy- 2.5 None 314 ± 49 5-nitropyrimidine(3e) 2.5 40  2 ± 1

Although the human alkyltransferase is very sensitive to inactivation byO⁶-benzylguanine and the various compounds described above, a number ofmutants have been generated that are resistant to O⁶-benzylguanine(Crone and Pegg, Cancer Res., 53, 4750-4753 (1993)). This resistance isprobably caused by a reduction in the space surrounding the active siteof the alkyltransferase, which limits the access to O⁶-benzylguanine;These mutants are produced by single base changes in thealkyltransferase DNA-coding sequence causing changes in one or two aminoacids in the alkyltransferase (Crone and Pegg, Cancer Res., 53,4750-4753 (1993)). Thus, as indicated in Table 6, changing the prolineresidue at position 140 to alanine (protein 2140A) or the glycineresidue at position 156 to an alanine (protein G156A) causes a 20-foldand a 240-fold increase in resistance to O⁶-benzylguanine, respectively.The alkyltransferase containing an arginine in place of a praline atresidue 138 together with an arginine in place of a praline at residue140 (protein P138A/P140A) is 88-fold more resistant to inactivation byO⁶-benzylguanine. It is possible that such resistant mutants will ariseor be selected for in tumors under the selective pressure generated bytreatment with O⁶-benzylguanine plus an alkylating agent. More potentinhibitors and/or those of a smaller size that are better able to fitinto the space of the active site of the mutant alkyltransferase can beused to advantage to overcome this resistance.

TABLE 6 Inhibition of Mutant Alkyltransferase Proteins byO⁶-Benzylguanine or 2,4-Diamino-6-benzyloxy-5-nitrosopyrimidine ED₅₀value (μM)^(a) 2,4-diamino-6- benzyloxy-5-nitroso- ProteinO⁶-benzylguanine pyrimidine Control 0.25 0.05 P140A 5 0.1 P138A/P140A 220.3 G156A 60 1 ^(a)The concentration needed to inactivate 50% of theactivity in 30 minutes.

As shown in Table 6, 2,4-diamino-6-benzyloxy-5-nitrasopyrimidine (3d)was 50 to 60 times better at inactivating the mutant alkyltransferasethan O⁶-benzylguanine. Doses of2,4-diamino-6-benzyloxy-5-nitrasopyrimidine leading to intracellularconcentrations greater than 5 μM will therefore be effective atinactivating such resistant alkyltransferase. Concentrations greaterthan 200 μM of O⁶-benzylguanine would be needed to get suchinactivation, and these are much more than can be achieved with thiscompound in current formulations. However, 8-substitutedO⁶-benzylguanine derivatives that are significantly more potent thanO⁶-benzylguanine may be useful in inactivating mutant alkyltransferaseprovided their required intracellular concentrations can be achieved.These data for mutant alkyltransferase inactivation and the datapresented earlier indicate that pyrimidine derivatives bearingelectron-withdrawing groups at the 5-position as well as substitutedO⁶-benzylguanine derivatives bearing electron-withdrawing groups at the8-position are superior to O⁶-benzylguanine for use as adjuvants inchemotherapy with agents whose mechanism of action, like that of BCNU,involves modification of the O⁶-position of DNA guanine residues.

Other 8-substituted O⁶-benzylguanine derivatives bearingelectron-withdrawing 8-substituents (e.g., NO₂) are readily available.For example, O⁶-benzyl-8-nitroguanine could be prepared by treatment of8-nitroguanine (Jones and Robins, J. Am. Chem. Soc., 82, 3773-3779(1960)) with phosphorus oxychloride to produce2-amino-6-chloro-8-nitropurine which when treated with sodiumbenzyloxide in benzyl alcohol would produce the desiredO⁶-benzyl-8-nitroguanine.

Additional 2,4-diamino-6-benzyloxypyrimidine derivatives bearingelectron-withdrawing groups other than halogen or nitro groups (e.g.,formyl or cyano groups) could also be readily prepared.2,4-Diamino-5-formyl-6-hydroxypyrimidine, a known compound (Delia andOtteman, Heterocycles, 20, 1805-1809 (1983)), can be treated withphosphorus oxychloride to produce a2,4-diamino-6-chloro-5-formylpyrimidine intermediate, which on treatmentwith sodium benzyloxide in benzyl alcohol produces2,4-diamino-6-benzyloxy-5-formylpyrimidine. Treatment of the formylpyrimidine with hydroxylamine affords2,4-diamino-6-benzyloxy-5-cyanopyrimidine. The preparation of a largenumber of 5-substituted 6(4)-benzyloxypyrimidines or 8-substitutedO⁶-benzylguanine derivatives is possible for those skilled in the art ofsynthesis of heterocyclic aromatic compounds (D. J. Brown, “ThePyrimidines,” in The Chemistry of Hetezogyclic Compounds, Vol. 16, A.Weissberger, Ed., Wiley Interscience, New York, 1962; D. J. Brown, “ThePyrimidines,” Supplement I, in The Chemistry of Heterocyclic Compounds,Vol. 16, A. Weissberger and E. C. Taylor,. Eds., Wiley Interscience, NewYork, 1970; J. H. Lister, “Fused Pyrimidines Part II Purines,” in TheChemistry of Heterocyclic Compounds, Vol. 24 Part II, A. Weissberger andE. C. Taylor, Eds., Wiley Interscience, New York, 1971).

Because many 9-substituted O⁶-benzylguanine derivatives exhibitexcellent AGT inactivation properties (Moschel et al., J. Med. Chear.,35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37, 342-347 (1994)),8,9-disubstituted analogs are expected to be similarly active.

These can be readily prepared by reacting the anion of 8-substitutedO⁶-benzylguanines (e.g., 1a-e) or the anion of 8-aza-O⁶-benzylguanine(2) with any of the range of compounds already described (Moschel etal., J. Med. Chem., 35, 4486-4491 (1992); Chae et al., J. Med. Chem.,37, 342-347 (1994)) to produce a mixture of isomeric 7,8- and8,9-disubstituted O⁶-benzylguanine derivatives. The desired8,9-disubstituted derivative can be isolated and purified by silica gelcolumn chromatography as already described (Moschel et al., J. Med.Chem., 35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37, 342-347(1994)). Compound 7 was prepared by treating the anion of compound 1ewith methyl iodide in N,N-dimethylformamide. Compounds 8-12 wereprepared using similar procedures.

The O⁶-substituted compounds of the present invention can beadministered in any suitable manner to a mammal for the purpose ofenhancing the chemotherapeutic treatment of a particular cancer.Although more than one route can be used to administer a particularcompound, a particular route can provide a more immediate and moreeffective reaction than another route. Accordingly, the describedmethods provided herein are merely exemplary and are in no way limiting.

Generally, the O⁶-substituted compounds of the present invention asdescribed above will be administered in a pharmaceutical composition toan individual afflicted with a cancer. Those undergoing or about toundergo chemotherapy can be treated with the O⁶-substituted compoundsseparately or in conjunction with other treatments, as appropriate. Intherapeutic applications, compositions are administered to a patient inan amount sufficient to elicit an effective depression of AGT activitythereby potentiating the cytotoxicity of the aforedescribedchemotherapeutic treatment. An amount adequate to accomplish this isdefined as a “therapeutically effective dose,” which is also an “AGTinactivating effective amount.” Amounts effective for a therapeutic orprophylactic use will depend on, e.g., the stage and severity of thedisease being treated, the age, weight, and general state of health ofthe patient, and the judgment of the prescribing physician. The size ofthe dose will also be determined by the O⁶-substituted compoundselected, method of administration, timing and frequency ofadministration as well as the existence, nature, and extent of anyadverse side-effects that might accompany the administration of aparticular O⁶-substituted compound and the desired physiological effect.It will be appreciated by one of skill in the art that various diseasestates may require prolonged treatment involving multipleadministrations, perhaps using a series of different AGT inactivatorsand/or chemotherapeutic agents in each or various rounds ofadministration.

Suitable chemotherapeutic agents usefully administered in coordinationwith the O⁶-substituted compounds of the present invention includealkylating agents, such as chloroethylating and methylating agents. Suchagents may be administered using conventional techniques such as thosedescribed in Wasserman et al., Cancer, 36, pp. 1258-1268 (1975), andPhysicians' Desk Reference, 48th ed., Edward R. Barnhart publisher(1994). For example, 1,3-bis(2-chloroethyl)-1-nitrosourea (carmustine orBCNU, Bristol-Myers, Evansville, Ind.) may be administered intravenouslyat a dosage of from about 150 to 200 mg/m² every six weeks. Anotheralkylating agent, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea(lomustine or CCNU, Bristol-Myers), may be administered orally at adosage of about 130 mg/m² every six weeks. Other alkylating agents maybe administered in appropriate dosages via appropriate routes ofadministration known to skilled medical practitioners.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages that are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. The present inventive method typically willinvolve the administration of about 0.1 mg to about 50 mg of one or moreof the compounds described above per kg body weight of the individual.For a 70 kg patient, dosages of from about 10 mg to about 200 mg ofO⁶-substituted compound would be more commonly used, possibly followedby further lesser dosages from about 1 mg to about 1 mg ofO⁶-substituted compound over weeks to months, depending on a patient'sphysiological response, as determined by measuring cancer-specificantigens or other measurable parameters related to the tumor load of apatient.

It must be kept in mind that the compounds and compositions of thepresent invention generally are employed in serious disease states, thatis, life-threatening or potentially life-threatening situations. In suchcases, in view of the minimization of extraneous substances and therelative nontoxic nature of the O⁶-substituted compounds, it is possibleand may be felt desirable by the treating physician to administersubstantial excesses of these O⁶-substituted compounds.

Single or multiple administrations of the compounds can be carried outwith dose levels and pattern being selected by the treating physician.In any event, the pharmaceutical formulations should provide a quantityof AGT-inactivating compounds of the invention sufficient to effectivelyenhance the cytotoxic impact of the chemotherapy.

The pharmaceutical compositions for therapeutic treatment are intendedfor parental, topical, oral or local administration and generallycomprise a pharmaceutically acceptable carrier and an amount of theactive ingredient sufficient to reduce, and preferably prevent, theactivity of the AGT protein. The carrier may be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with thecompound, and by the route of administration.

Examples of pharmaceutically-acceptable acid addition salts for use inthe present inventive pharmaceutical composition include those derivedfrom mineral acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric and sulfuric acids, and organic acids, such astartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,gluconic, succinic, p-toluenesulphonic acids, and arylsulphanic, forexample.

The pharmaceutically acceptable excipients described herein, forexample, vehicles, adjuvants, carriers or diluents, are well-known tothose who are skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone that is chemically inert to the active compounds and one that has nodetrimental side effects or toxicity under the conditions of use. Suchpharmaceutically acceptable excipients preferably include saline (e.g.,0.9% saline), Cremophor EL (which is a derivative of castor oil andethylene oxide available from Sigma Chemical Co., St. Louis, Mo.) (e.g.,5% Cremophor EL/5% ethanol/90% saline, 10% Cremophor EL/90% saline, or50% Cremophor EL/50% ethanol), propylene glycol (e.g., 40% propyleneglycol/10% ethanol/50% water), polyethylene glycol (e.g., 40% PEG400/60% saline), and alcohol (e.g., 40% t-butanol/60% water). The mostpreferred pharmaceutical excipient for use in conjunction with thepresent invention is polyethylene glycol, such as PEG 400, andparticularly a composition comprising 40% PEG 400 and 60% water orsaline.

The choice of excipient will be determined in part by the particularO⁶-substituted compound chosen, as well as by the particular method usedto administer the comparison. Accordingly, there is a wide variety ofsuitable formulations of the pharmaceutical composition of the presentinvention.

The following formulations for oral, aerosol, parenteral, subcutaneous,intravenous, intraarterial, intramuscular, interperitoneal, rectal, andvaginal administration are merely exemplary and are in no way limiting.

The pharmaceutical compositions can be administered parenterally, e.g.,intravenously, intraarterially, subcutaneously, intradermally, orintramuscularly. Thus, the invention provides compositions forparenteral administration that comprise a solution of the O⁶-substitutedcompound dissolved or suspended in an acceptable carrier suitable forparenteral administration, including aqueous and non-aqueous, isotonicsterile injection solutions.

Overall, the requirements for effective pharmaceutical carriers forparenteral compositions are well known to those of ordinary skill in theart. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986). Such solutions can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The compound may be administeredin a physiologically acceptable diluent in a pharmaceutical carrier,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol, isopropanol, or hexadecyl alcohol, glycols, such as propyleneglycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, suchas 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils useful in parenteral formulations include petroleum, animal,vegetable, or synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-b-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations typically will contain from about 0.5% toabout 25% by weight of the active ingredient in solution. Preservativesand buffers may be used. In order to minimize or eliminate irritation atthe site of injection, such compositions may contain one or morenonionic surfactants having a hydrophile-lipophile balance (HLB) of fromabout 12 to about 17. The quantity of surfactant in such formulationswill typically range from about 5% to about 15% by weight. Suitablesurfactants include polyethylene sorbitan fatty acid esters, such assorbitan monooleate and the high molecular weight adducts of ethyleneoxide with a hydrophobic base, formed by the condensation of propyleneoxide with propylene glycol. The parenteral formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

Topical formulations, including those that are useful for transdermaldrug release, are well-known to those of skill in the art and aresuitable in the context of the present invention for application toskin.

Formulations suitable for oral administration require extraconsiderations considering the peptidyl and/or carbohydrate nature ofsome of the O⁶-substituted compounds of the present invention and thelikely breakdown thereof if such compounds are administered orallywithout protecting them from the digestive secretions of thegastrointestinal tract. Such a formulation can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant, suspending agent, or emulsifying agent. Capsule forms can beof the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and corn starch. Tablet forms can includeone or more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible excipients. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin, or sucrose and acacia, emulsions, gels,and the like containing, in addition to the active ingredient, suchexcipients as are known in the art.

The O⁶-substituted compounds of the present invention, alone or incombination with other suitable components, can be made into aerosolformulations to be administered via inhalation. The compounds arepreferably supplied in finely divided form along with a surfactant andpropellant. Typical percentages of active compound are 0.01-20% byweight, preferably 1%-10%. The surfactant must, of course, be nontoxic,and preferably soluble in the propellant. Representative of suchsurfactants are the esters or partial esters of fatty acids containingfrom 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural glycerides may be employed. The surfactant mayconstitute 0.1%-20% by weight of the composition, preferably 0.25-5%.The balance of the composition is ordinarily propellant. A carrier canalso be included as desired, e.g., lecithin for intranasal delivery.These aerosol formulations can be placed into acceptable pressurizedpropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations may be used to spray mucosa.

Additionally, the compounds and polymers useful in the present inventivemethods may be made into suppositories by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

The concentration of the O⁶-substituted compounds of the presentinvention in the pharmaceutical formulations can vary widely, i.e., fromless than about 1%, usually at or at least about 10%, to as much as 20%to 50% or more by weight, and will be selected primarily by fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 100mg of the O⁶-substituted compound. Actual methods for preparingparenterally administrable compounds will be known or apparent to thoseskilled in the art and are described in more detail in, for example,Remington's Pharmaceutical Science (17th ed., Mack Publishing Company,Easton, Pa., 1985).

It will be appreciated by one of ordinary skill in the art that, inaddition to the aforedescribed pharmaceutical compositions, theO⁶-substituted compounds of the present inventive method may beformulated as inclusion complexes, such as cyclodextrin inclusioncomplexes, or liposomes. Liposomes serve to target the compounds to aparticular tissue, such as lymphoid tissue or cancerous hepatic cells.Liposomes can also be used to increase the half-life of theO⁶-substituted compound. Liposomes useful in the present inventioninclude emulsions, foams, micelles, insoluble monolayers, liquidcrystals, phospholipid dispersions, lamellar layers and the like. Inthese preparations, the O⁶-substituted compound to be delivered isincorporated as part of a liposome, alone or in conjunction with asuitable chemotherapeutic agent. Thus, liposomes filled with a desiredO⁶-substituted compound of the invention can be directed to the site ofa specific tissue type, hepatic cells, for example, where the liposomesthen deliver the selected chemotherapeutic-enhancement compositions.Liposomes for use in the invention are formed from standardvesicle-forminq lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of, for example, liposomesize and stability of the liposomes in the blood stream. A variety ofmethods are available for preparing liposomes, as described in, forexample, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980), andU.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. Fortargeting to the cells of a particular tissue type, a ligand to beincorporated into the liposome can include, for example, antibodies orfragments thereof specific for cell surface determinants of the targetedtissue type. A liposome suspension containing an O⁶-substituted compoundmay be administered intravenously, locally, topically, etc. in a dosethat varies according to the mode of administration, the O⁶-substitutedcompound being delivered, the stage of disease being treated, etc.

While the efficacy of the O⁶-substituted compounds of the presentinvention has been demonstrated with respect to particular types ofcancerous cells, e.g., colon, prostate, and breast cancer cells, thepresent invention has applicability to the treatment of any type ofcancer capable of being treated with an antineoplastic alkylating agentwhich causes cytotoxic lesions at the O⁶-position of guanine. Suchcancers include, for example, colon tumors, prostrate tumors, braintumorsL lymphomas, leukemias, breast tumors, ovarian tumors, lungtumors, Wilms' tumor, rhabdomyosarcoma, multiplemyeloma, stomach tumors,soft-tissue sarcomas, Hodgkin's disease, and non-Hodgkin's lymphomas.

Similarly, in view of the mode of action of the O⁶-substituted compoundsof the present invention, such compounds can be used in conjunction withany type of antineoplastic alkylating agent which causes cytotoxiclesions at the O⁶-position of guanine. Such antineoplastic alkylatingagents include, for example, chloroethylating agents (e.g.chloroethylnitrosoureas and chloroethyltriazines) and monofunctionalalkylating agents such as Streptozotocin, Procarbazine, Dacarbazine, andTemozolomide.

Among the chloroethylating agents, the most frequently usedchemotherapeutic drugs are 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea(CCNU, lomustLne), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU,carmustine), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea(MeCCNU, semustine), and1-(2-chloroethyl)-3-(4-amino-2-methyl-S-pyrimidinyl)methyl-1-nitrosourea(ACNU). These agents have been used clinically against tumors of thecentral nervous system, multiple myeloma, melanoma, lymphoma,gastrointestinal tumors, and other solid tumors (Colvin and Chabner,Alkylating Agents. In: Cancer Chemotherapy: Principles and Practice,Chabner and Collins, eds., Lippincott, Philadelphia, pp. 276-313 (1990);McCormick and McElhinney, Eur. J. Cancer, 26, 207-221 (1990)).Chloroethylating agents currently under development with fewer sideeffects are 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea (HECNU),2-chloroethyl-methylsulfonylmethanesulfonate (Clomesone), and1-[N-(2-chloroethyl)-N-nitrosoureido]ethylphosphonic acid diethyl ester(Fotemustine) (Colvin and Chabner, Alkylating Agents. In: CancerChemotherapy: Principles and Practice, Chabner and Collins, eds.,Lippincott, Philadelphia, pp. 276-313 (1990); McCormick and McElhinney,Eur. J. Cancer, 26, 207-221 (1990)). Methylating chemotherapeutic agentsinclude Streptozotocin(2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranose), Procarbazine(N-(1-methylethyl)-4-[(2-methylhydrazino)methyl]benzamide), Dacarbazineor DTIC (5-(3,3-dimethyl-1-triazenyl) -1H-imidazole-4-carboxamide), andTemozolomide(8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazine-4-(3H)-one).Temozolomide is active against malignant melanomas, brain tumors, andmycosis fungoides. Streptozotocin is effective against pancreatictumors. Procarbazine is used to treat Hodgkin's disease and braintumors, and DTIC is used in treatment of melanoma and lymphomas (Colvinand Cabner, Alkylating Agents. In: Cancer Chemotherapy: Principles andPractice, Chabner and Collins, eds., Lippincott, Philadelphia, pp.276-313 (1990); Longo, Semin. Concol., 17, 716-735 (1990)).

The examples set forth below describe the syntheses of theaforedescribed compounds. As regards the methods and materials set forthin these examples, ¹H—NMR spectra were recorded on a Varian VXR 500Sspectrometer equipped with Sun 2/110 data stations or a Varian XL 200instrument interfaced to an Advanced data system. Samples were dissolvedin DMSO-d₆ with Me₄Si as an internal standard. EI mass spectra wereobtained on a reversed geometry VG Micromass ZAB-2F spectrometerinterfaced to a VG 2035 data system. Elemental analyses were performedby Galbraith Laboratories, Inc., Knoxville, Tenn.

Most of the reagents and solvents were from Aldrich Chemical Co., Inc.,Milwaukee, Wis. 8-Aza-O⁶-benzylguanine (2) (Shealy et al., J. Org.Chem., 27, 4518-4523 (1962)), 2, 4-diamino-6-benzyloxypyrimidine (3b)(Pfleiderer and Lohzmann, Chem. Ber., 94, 12-18 (1961)),2,4,5-triamino-6-benzyloxypyrimidine (3c) (Pfleiderer and Lohrmann,Chem. Ber., 94, 12-18 (1961)),2,4-diamino-6-benzyloxy-5-nitrasopyrimidine (3d) (Pfleiderer andLohrman, Chem. Ber., 94, 12-18 (1961)),2,4-diamino-6-benzyloxy-5-nitropyrimidine (3e) (Kosary et al., ActaPharm. Hung. 49, 241-247 (1989)),2,4-diamino-6-benzyloxy-5-bromapyrimidine (3f) (Kosary et al., ActaPhazm. Hung., 49, 241-247 (1989)), 4-amino-6-benzyloxy-5-nitropyrimidine(3a) and O⁶-benzyl-2-fluorohypoxanthine (4c) (Robins and Robins, J. Org.Chem., 34, 2160-2163 (1969)) were prepared previously. Alternativesynthetic methods are provided below for some of these compoundstogether with spectroscopic data not provided previously. AGTinactivation studies were carried out as described in Moschel et al., J.Med. Chem., 35, 4486-4491 (1992). Cell killing experiments involvingvarious AGT inactivators in combination with BCNU were carried out as inDolan, et al. (Proc. Natl. Acad. Sci. U.S.A., 87; 5368-5372 (1990)).Cells were treated for 2 h with AGT inactivator prior to exposure toBCNU.

EXAMPLE 1

2,8-Diamino-6-chloropurine

A suspension of 8-aminoguanine (Fischer, Z. Physiol. Chem., 60, 69(1909); Beaman et al., in Zorbach and Tipson, Synthetic Procedures inNucleic Acid Chemistry, Vol. 1, pp 41-43, John Wiley & Sons, New York,1968) (3.0 g, 18.1 mmol) in phosphorus oxychloride (90 mL) andN,N-diethylaniline (3 mL) was refluxed for 30 min and the excessphosphorus oxychloride was evaporated under reduced pressure. Ice (20 g)was added slowly to the resulting solution and the pH was adjusted to 6with a concentrated aqueous sodium hydroxide solution. A yellow solidformed and was collected by filtration, washed with water, and dried togive a green solid. Crystallization from water with charcoal treatmentproduced 2,8-diamino-6-chloropurine as a white solid: yield, 2.11 g(63%); mp >275° C. dec.; ¹H NMR d 6.09 (s, 2 H, NH₂, exchange with D₂O),6.71 (s, 2 H, NH₂, exchange with D₂O); MS (EI) calcd. m/z for C₅H₅N₆³⁵Cl 184.0264, found 184.0266; calcd. m/z C₅H₅N₆ ³⁷Cl 186.0235, found186.0237.

EXAMPLE 2

8-Amino-O⁶-benzylguanine (1a)

2,8-Diamino-6-chloropurine (0.9 g, 4.9 mmol) was added to the solutionof sodium (0.22 g, 10 mmol) in benzyl alcohol (9.0 mL). The solution washeated in a 130° C. oil bath for 5 h, and was poured into water (100 mL)with constant stirring for 10 min. Undissolved solid was removed byfiltration and the filtrate was neutralized with glacial acetic acid.The solution was mixed with methanol (100 mL), and half of the aqueousmethanol solution was loaded an a 3×80 cm Sephadex LH-20 column elutedwith methanol/water (1:1) at 1 mL/min. Column eluent was continuouslymonitored at 280 nm and fractions (10 mL) were collected. The remainderof the reaction mixture in MeOH/H₂O was chromatographed separately underidentical conditions. The desired product eluted in fractions 100-130.Evaporation of solvent from the pooled fractions 100-130 from bothchromatographic runs afforded analytically pure 1a: yield, 0.26 g (21%);mp 269-271° C. dec.; UV (pH 1) 1_(max) 241 nm (e=0.699×10⁴), 300(1.109×10⁴); (pH 6.9) 250 (sh) (0.447×10⁴), 292 (1.027×10⁴) ; (pH 13)255 (sh) (0.355×10⁴), 295 (0.932×10⁴); ¹H NMR d 5.41 (s, 2 H, ArCH₂),5.70 (S, 2 H, NH₂, exchange with D₂O), 6.18 (s, 2 H, NH₂, exchange withD₂O), 7.25-7.55 (m, 5 H, ArH), 11.1 (br s, 1 H, NH, exchanges with D₂O); MS (EI) calcd. m/z for C₁₂H₁₂N₆O 256.1072, found 256.1059; Anal.(CH₁₂H₁₂N₆O) C, H, N.

EXAMPLE 3

2-Amino-6-chloro-8-methylpurine

A suspension of 8-methylguanine (Daves et al., J. Am. Chem. Soc., 82,2633-2640 (1960)) (1.0 g, 6.1 mmol) in phosphorous oxychloride (30 mL)and N,N-diethylaniline (1 mL) was refluxed for 3 h. The excessphosphorous oxychloride was evaporated under reduced pressure. Theresulting brown oil was dissolved in ice-water and was neutralized witha concentrated aqueous NaOH solution.

After evaporation of the solvent, the solid residue was suspended in 70mL of H₂O. Undissolved solid was filtered off, and the filtrate wasloaded an a 3×60 cm Sephadex LH-20 column eluted with methanol/water(1:1) at 1 mL/min. Column eluent was continuously monitored at 280 nmand fractions (10 mL) were collected. Evaporation of pooled fractions50-60 produced 2-amino-6-chloro-8-methylpurine as a crude solid.Crystallization from ethanol/water with charcoal treatment afforded2-amino-6-chloro-8-methylpurine as a white solid: yield, 0.57 g(51%);mp >265° C. dec.; ¹H NMR d 2.39 (s, 3 H, CH₃), 6.62 (s, 2 H, NH₂,exchange with D₂O), 12.56 (s, 1 H, NH, exchanges with D₂O); MS (EI)calcd. m/z for C₆H₆N₅ ³⁵Cl 183.0312, found 183.0309; calcd. m/z forC₆H₆N₅ ³⁷Cl 185.0283, found 185.0286.

EXAMPLE 4

O⁶Benzyl-8-methylguanine (1b)

Sodium (0.1 g, 4.4 mmol) was stirred in 4.1 mL of benzyl alcohol untilall sodium had reacted. 2-Amino-6-chloro-8-methylpurine (0.41 g, 2.2mmol) was added, and the reaction mixture was heated in a 130° C. oilbath ror 5 h. After cooling to room temperature 40 mL of ether was addedto remove excess benzyL alcohol. The sticky precipitate that formed wascollected by filtration and was dissolved in water (50 mL). The pH ofthe yellow solution was adjusted to 5-6 with glacial acetic acid. Thesolution was mixed with methanol (50 mL) and was loaded on a 3×80 cmSephadex LH-20 column eluted with methanol/water (1:1) at 1 mL/min.Column eluent was continuously monitored at 280 nm and fractions (10 mL)were collected. Evaporation of pooled fractions 78-93 affordedanalytically pure 1b: yield, 0.25 g (44%); mp 214-216° C.; UV (pH 1)1_(max) 238 nm (sh) (e=0.648×10⁴), 290 (1.136×10⁴) ; (pH 6.9) 242(0.758×10⁴), 284 (0.897×10⁴); (pH 13) 240 (sh) (0.495×10⁴) 286(0.932×10⁴); ¹H NMR d 2.33 (s, 3 H, CH₃), 5.46 (s, 2 H, ArCH₂), 6.17 (s,2 H, NH₂, exchange with D₂O),7.34-7.51 (m, 5 H, ArH), 12.18 (br s, 1 H,NH, exchanges with D₂O),; MS (EI) calcd. m/z for C₁₃H₁₃N₅O 255.1120,found 255.1125; Anal. (C₁₃H₁₃N₅O. ¼ H₂O) C, H, N.

EXAMPLE 5

O⁶-Benzyl-8-oxoguanine (1c)

2,4,5-Triamino-6-benzyloxypyrimidine (Pfleiderer et al., Chem. Ber., 94,12-18 (1961)) (1.85 g, 8 mmol) and 1,1′-carbonyldiimidazole (1.30 g, 8mmol) were dissolved in anhydrous N,N-dimethvlformamide (5 mL) underargon. The solution was stirred at room temperature overnight and wasmixed with water (200 mL) to precipitate a white solid. The solid wascollected by filtration, and dissolved in 250 mL of aqueous 2 N NaOHsolution. Undissolved material was removed by filtration, and thefiltrate was neutralized with glacial acetic acid to precipitate a whitesolid. The solid was collected by filtration, was washed with water, andwas recrystallized from 50% aqueous ethanol to afford analytically pure1c: yield, 1.63 g (79%); mp 256-257° C. dec.; UV (pH 1) 1_(max) 243 nm(e=0.717×10⁴), 306 (1.499×10⁴); (pH 6.9) 243 (0.915×10⁴), 290(1.108×10⁴); (pH 13) 249 (sh) (0.443×10⁴), 293 (1.368×10⁴); 1H NMR d5.41 (s, 2 H, ArCH₂), 6.13 (s, 2 H, NH₂, exchange with D₂O), 7.33-7.51(m, 5 H, ArH), 10.46 (s, 1 H, exchanges with D₂O), 11.04 (s, 1 H,exchanges with D₂O); MS (ET) Calcd. m/z for C₁₂H₁₁N₅O₂:257.0912. Found:257.0914. Anal. (C₁₂H₁₁N₅O₂. ½ H₂O) C, N, H.

EXAMPLE 6

O⁶-Benzyl-8-bromoguanine (1d)

Bromine (0.26 mL, 5.1 mmol) was added slowly to the solution ofO⁶-benzylguanine (1.205 g, 5.0 mmol) in anhydrous DMF (10 mL) underargon. The resulting deep green solution was stirred at room temperatureovernight. The solution was mixed with water (70 mL) to precipitatecrude product. This product was collected by filtration and wasdissolved in 50% aqueous methanol (100 mL). The solution was loaded on a3×80 cm Sephadex LH-20 column eluted with methanol/water (1:1) at 1mL/min. Column eluent was continuously monitored at 280 nm and fractions(10 mL) were collected. The desired product eluted in fractions 110-190.Evaporation of solvent from the pooled fractions 110-190 afforded Id asa pale yellow solid. Crystallization from ethanol/water (1:1) producedanalytically pure lid: yield, 0.166 g (10%); mp 135-137° C. dec.; UV(pH 1) 1_(max) 236 nm (sh) (e=0.517×10⁴), 294 (1.429×10⁴); (pH 6.9) 244(0.666×10⁴), 287 (1.043×10⁴); (pH 13) 245 (sh) (0.544×10⁴), 289(1.030×10⁴); ¹H NMR d 5.45 (s, 2 H, ArCHz), 6.35 (s, 2, H, NH₂, exchangewith D₂O), 7.34-7.52 (m, 5 H, ArH), 13.08 (b s, 1 H, NH, exchanges withD₂O) ; MS (EI) calcd. m/z for C₁₂H₁₀N₅O⁷⁹Br 319.0068, found 319.0069;calcd. m/z for C₁₂H₁₀N₅O⁸¹ Br 321.0048, found 321.0048; Anal.(C₁₂H₁₀N₅OBR{fraction (3/2)} H₂O) C, H, N, Br.

EXAMPLE 7

8-Aza-O⁶-benzylguanine (2)

Glacial acetic acid (1 mL) was added into the mixture of2,4,5-triamino-6-benzyloxypyrimidine (0.231 g, 1.0 mmol) and sodiumnitrite (0.069 g, 1.0 mmol) in acetone (5 ml). The resulting mixture wasstirred at room temperature for 2 h. The solution was poured in water(100 mL) with stirring to precipitate a crude solid. The solid wascollected by filtration and air dried. Crystallization fromethanol/water (1:1) with charcoal treatment produced 2 as a white solid:yield, 105 mg (43%); mp 191-192° C. (192-193° C.; Shealy et. al., J.Org. Chem., 27, 4518-4523 (1962)); ¹H NMR d 5.56 (s, 2 H, ArCH₂), 7.00(s, 2 H, NH₂, exchange with D₂O), 7.41-7.58 (m, 5 H, ArH) ; MS (EI)calcd. m/z for C₁₁H₁₀N₆O 242.0916, found 242.0924.

EXAMPLE 8

4-Amino-6-benzyloxy-5-nitropyrimidine (3a)

4-sAmino-6-chloro-5-nitropyrimidine (Boon et al., J. Chem. Soc., 96-102(1951)) (1.5 g, 8.6 mmol) was added to a solution of sodium (0.23 g, 9.9mmol) in benzyl alcohol (14 mL). The solution was heated in a 130° C.oil bath for 3.5 h, and was poured into benzene (50 mL). A yellow solidwas collected by filtration and washed with benzene. Crystallizationfrom benzene/ether afforded an analytically pure sample of 3a: yield,0.71 g (34%); mp 149-150° C.; UV (pH 1) 1_(max) 284 nm (e=0.368×10⁴),333 (0.488×10⁴); (pH 6.9) 284 (0.329×10⁴), 336 (0.470×10⁴); (pH 13) 290(0.344×10⁴) 333 (0.494×10⁴); ¹H NMR d 5.50 (s, 2 H, ArCH₂), 7.33-7.49(m, 5 H, ArH), 8.12-8.24 (br d, 2 H, NH_(a) and NE_(b), exchange withD₂O), 8.24 (s, 1 H, H-2); MS (EI) calcd. m/z for C₁₁H₁₀N₄O₃ 246.0752,found 246.0751; Anal. (C₁₁H₁₀N₄O₃) C, H, N.

EXAMPLE 9

2,4-Diamina-6-benzyloxy-5-nitropyrimidine (3e)

2,4-Diamino-6-chloro-5-nitropyrimidine (O'Brien et. al., J. Med. Chem.,9, 573-575 (1966)) (1.0 g, 5.28 mmol) was added to a solution of sodium(0.14 g, 6.08 =mol) in benzyl alcohol (9 mL). The solution was heated ina 160° C. oil bath for 3.5 h and the solvent was evaporated underreduced pressure to provide a yellow solid. This solid was washed withwater, and air dried. Crystallization from benzene/ether gave a paleyellow filamentous solid: yield, 0.69 g (50%); mp 194-195° C. (171° C.;Kosary et. al., Acta. Pharm. Hung., 49, 241-247 (1989)); UV (pH 1)1_(max) 236 nm (sh) (e=1.452×10⁴) 264 (0.522×10⁴) 321 (1.294×10⁴) ; (pH6.9) 242 (sh) (0.965×10⁴), 337 (1.493×10⁴); (pH 13) 242 (sh)(0.952×10⁴), 338 (1.479×10⁴); ¹H NMR d 5.43 (s, 2 H, ArCH₂), 7.26 (br s,2 H, NH₂, exchange with D₂O), 7.33-7.51 (m, 5 H, ArH), 7.93 (br s, 2 H,NH₂, exchange with D₂O); MS (EI) calcd. m/z for C₁₁H₁₁N₅O₃ 261.0861,found 261.0866; Anal. (C₁₁H₁₁N₅O₃).

EXAMPLE 10

O⁶-Benzylxanthine (4a)

A suspension of O⁶-benzylguanine (0.83 g, 3.4 mmol) in acetone (15 mL)was poured into a solution of sodium nitrite (5 g) in 15 mL of H₂O.Acetic acid (8 mL) was added to the suspension with stirring. Minimumamounts of acetone were added as necessary to dissolve any suspendedsolid. The resulting pale yellow-green solution was stirred for 3 h. Apale green precipitate that formed was collected by filtration andwashed with water (200 mL). Recrystallization of the air-dried solidfrom ethanol/water (1:1) afforded analytically pure 4a: yield, 0.43 g(52%); mp 145-147° C. dec.; UV (pH 1) 1_(max) 270 nm (e=0.749×10⁴); (pH6.9) 286 (1.143×10⁴); (pH 13) 290 (0.914×10⁴); ¹H NMR d 5.49 (s, 2 H,ArCH₂), 7.36-7.54 (m, 5 H, ArH), 8.02 (s, 1 H, H-8), 11.8 (br s, 1 H,NH, exchanges with D₂O), 13.2 (br s, 1 H, NH, exchanges with D₂O); MS(EI) calcd. m/z for C₁₂H₁₀N₄O₂ 242.0803, found 242.0828; Anal.(C₁₂H₁₀N₄O₂. H₂O) C, H, N.

EXAMPLE 11

O⁶-Benzyluric acid (4b)

Sodium nitrite (1.5 g, 43 mmol) dissolved in water (5 mL) was added to asuspension of O⁶-benzyl-8-oxoguanine (1c) (0.257 g, 1.0 mmol) in acetone(5 mL). Glacial acetic acid (3 mL) was added to the suspension withstirring. After stirring for 3 h at room temperature a bright yellowprecipitate formed. The suspension was mixed with water (150 mL) andundissolved solid was filtered off. Saturated aqueous sodium carbonatesolution was added to the filtrate to adjust the pH to approximately 5.A yellow precipitate (130 mg) was collected and washed with water. Thissolid was crystallized from 50% aqueous ethanol to give an analyticallypure sample of 4b: yield, 75 mg (29%); mp >230° C.; UV (pH 1) 1_(max)236 nm (sh) (e=0.972×10⁴), 299 (1.427×10⁴); (pH 6.9) 240 (sh)(0.821×10⁴), 304 (2.134×10⁴); (pH 13) 245 (sh) (0.846×10⁴), 297(1.861×10⁴); ¹H NMR d 5.43 (s; 2 H, ArCH₂), 7.35-7.51 (m, 5 H, ArH),10.76 (s, 1 H, NH, exchanges with D₂O), 11.23-(s, 1 H, NH, exchangeswith D₂O), 11.39 (s, 1 H, NH, exchanges with D₂O); MS (EI) calcd. m/zfor C₁₂H₁₀N₄O₃ 258.0752, found 258.0753; Anal. (C₁₂H₁₀N₄O₃.{fraction(5/2)} H₂O) C, H, N.

EXAMPLE 12

Diacetyl-O⁶-benzyl-8-oxoguanine

Acetic anhydride (2 mL) was added to the suspension ofO⁶-benzyl-8-oxoguanine (1c) (0.257 g, 1.0 mmol) in dry toluene (10 mL).The suspension was vigorously refluxed for 24 hr, and was cooled to roomtemperature. After storing at 4° C. for 4 hr, the resulting precipitatewas collected by filtration, washed with benzene and air dried to givean analytically pure sample of a diacetylated product: yield, 0.287 g(84%); mp 272-274° C. dec.; UV (100% MeOH) 1_(max) 275 nm (e=1.313×10⁴);(pH 1) 275 (1.143×10⁴); (pH 6.9) 238 (0.995×10⁴), 276 (1.115×10⁴); (pH13) 285 (2.138×10⁴); ¹H NMR d 2.18 (s, 3 H, CH₃), 2.57 (s, 3 H, CH₃),5.51 (s, 2 H, ArCH₂), 7.30-7.57 (m, 5 H, ArH), 10.41 (s, 1 H, exchangeswith D₂O), 12.30 (s, 1 H, exchanges with D₂O); MS (EI) Calcd. m/z forC₁₆H₁₅N₅O₄:341.1123. Found: 341.1130. Anal. (C₁₆H₁₅N₅O₄) C, N, H.

EXAMPLE 13

N²-Acetyl-O⁶-benzyl-8-oxoguanine (4d)

Diacetyl-O⁶-benzyl-8-oxoguanine (85 mg, 0.25 mmol) was dissolved inmethanol (10 mL) and ammonium hydroxide (28%, 5 mL) and was allowedstand for 1 hr. The clear solution became cloudy and a precipitateformed on standing. The precipitate was collected by filtration, washedwith water, and dried to give an analytically pure sample of 4d: yield,48 mg (65%); mp 335-337° C. dec.; UV (pH 1) 1_(max) 276 nm(e=1.723×10⁴), 303 (sh) (0.679×10⁴); (pH 6.9) 276 (1.379×10⁴); (pH 13)284 (1.683×10⁴); ¹H NMR d 2.15 (s, 3 H, CH₃), 5.49 (s, 2 H, ArCH₂),7.30-7.55 (m, 5 H, ArH), 10.21 (s, 1 H, exchanges with D₂O), 10.99 (s, 1H, exchanges with D₂O), 11.60 (s, 1 H, exchanges with D₂O; MS (EI)Calcd. m/z for C₁₄H₁₃N₅O₃:299.1018. Found: 299.1023. Anal. (C₁₄H₁₃N₅O₃)C, N, H.

EXAMPLE 14

O⁶-Benzyl-2-fluorchypoxanthine (4c)

O⁶-Benzylguanine (1.21 g, 5 mmol) was added to 100 mL of 48% fluoboricacid at −20° C. Sodium nitrite (1.23 g, 35 mmole) was dissolved in water(5 mL) and 2.5 mL of this sodium nitrite solution was added slowly tothe cold fluoboric acid solution. The resulting mixture was stirred for1 h at or below −15° C. Additional fluoboric acid (25 mL) was addedfollowed by an additional 2.5 mL of the aqueous sodium nitrite solution.After stirring for an additional 1 h below −15° C., fluoboric acid (25mL) was again added and stirring was continued for 1 h. The resultingsolution was neutralized with saturated aqueous sodium carbonatesolution at −20° C. and was allowed to warm to room temperature. A whiteprecipitate that formed was collected by filtration and was washed withwater and dried under vacuum to afford crude 4c: yield, 0.52 g, 43%. Ananalytical sample was prepared by chromatography on a Sephadex LH-20column (3×80 cm) eluted with methanol/water (1:1) at 1 mL/min. Thedesired4c eluted in fractions 66-77: up 182-183° C. (184-185° C.; Robinsand Robins, J. Org. Chem., 34, 2160-2163 (1969)); UV (pH 1) 1_(max) 256nm (e=1.117×10⁴); (pH 6.9) 257 (1.078×10⁴) (pH 13) 264 (1.063×10⁴); ¹HNMR d 5.60 (s, 2 H, ArCH₂), 7.37-7.57 (m, 5 H, ArH), 8.40 (s, 1 H, H-8),13.60 (s, 1 H, NH, exchanges with D₂O), ¹⁹F NMR d 23.54 downfield fromtrifluoroacetic acid standard; MS (EI) calcd. m/z for CH₁₉H₉FN₄O244.0760, found 244.0756; Anal. (C₁₂H₉FN₄O.⅔ H₂O) C, H, N.

EXAMPLE 15

O⁶-Benzyl-N²-methylguanine (4e)

Fluoboric acid (48%, 30 mL) was cooled to −20° C. in an dry ice-acetonebath. 06-Benzylguanine (0.362 g, 1.5 mmol) was added with stirring.Sodium nitrite (0.369 g, 10.5 mmol) was dissolved in water (1 mL) and0.5 mL of this solution was added slowly to the cold fluoboric acidsolution. The resulting solution was stirred at or below −15° C. for 1h. More fluoboric acid (5 mL) was then added followed by 0.5 mL of thesodium nitrite solution. After stirring for 1 h at or below −15° C.,fluoboric acid (5 mL) was again added and stirring was continued for anadditional 1 h. Methylamine (40% in water, 60 mL) was then added at −20°C., and the resulting basic solution was stirred at room temperature for2 days. The solvent was evaporated under reduced pressure to produce awhite solid. The solid was suspended in 50 mL of H₂O with stirring for10 min. Undissolved material was collected by filtration and washed withwater. This solid was dissolved in 40 mL methanol/water (1:1) to whichwas added 1.2 mL of 28% aqueous ammonia solution. The solution wasloaded on a 3×80 cm Sephadex LH-20 column eluted with MeOH/H₂O/NH₄OH(30:70:3) at 1 mL/min. Column eluent was continuously monitored at 280nm and fractions (10 mL) were collected. Evaporation of the pooledfractions 106-127 gave an analytically pure sample of 4e: yield, 85 mg(22%); mp 189 −190° C.; UV (pH 1) 1_(max) 238 nm (sh) (e=0.665×10⁴), 297(0.904×10⁴); (pH 6.9) 246 (0.898×10⁴), 290 (0.676×10⁴); (pH 13) 240 (sh)(0.615×10⁴), 294 (0.674×10⁴); ¹H NMR d 2.30 (d, 3 H, CH₃), 5.50 (s, 2 H,ArCH), 6.75 (m, 1 H, MeNH, exchanges with D₂O), 7.31-7.53 (m, 5 H, ArH),7.82 (s, 1 H, H-8), 12.53 (s, 1 H, NH, exchanges with D₂O); MS (EI)calcd. m/z for C₁₃H₁₃N₅O 255.1120, found 255.1107; Anal. (C₁₃H₁₃N₅O.½H₂O) C, H, N.

EXAMPLE 16

O⁶-Benzyl-N²,N²-dimethylguanine (4f)

Fluoboric acid (48%, 40 mL) was cooled to −20° C. in an dry ice-acetonebath. O⁶-Benzylguanine (0.482 g, 2.0 mmol) was added with stirring.Sodium nitrite (0.492 g, 14.0 mmol) was dissolved in water (2 mL) and 1mL of this solution was added slowly to the cold fluoboric acidsolution. The resulting solution was stirred at or below −15° C. tor 1h. More fluoboric acid (10 mL) was added followed by the addition of 1mL of the sodium nitrite solution. After stirring for 1 h at or below−15° C., additional fluoboric acid (10 mL) was added with stirring for 1h. Dimethylamine (40% in water, 60 mL) was then added to the solution at−20° C., and the resulting mixture was allowed to warm to roomtemperature. The suspension became a clear solution and a precipitateformed within 10 min. After standing overnight at room temperature theprecipitate was collected by filtration and was washed with water. Thesolid was crystallized from 50% aqueous ethanol to give an analyticallypure sample of 4f: yield, 0.25 g (46%); mp 220-221° C. dec.; UV (pH 1)1_(max) 248 nm (sh) (e=0.512×10⁴), 303 (0.908×10⁴); (pH 6.9) 251(1.152×10⁴) 299 (0.686×10⁴); (pH 13) 248 (sh) (0.766×10⁴) 299(0.710×10⁴); ¹H NMR d 3.12 (s, 6 H, CH₃), 5.54 (s, 2 H, ArCH₂),7.36-7.51 (m, 5 H, ArH), 7.84 (s, 1 H, H-8), 12.56 (s, 1 H, NH,exchanges with D₂O); MS (EI) calcd. m/z for C₁₄H₁₅N₅O 269.1276, found269.1254; Anal. (C₁₄H₁₅N₅O) C, H, N.

EXAMPLE 17

2,4-Diamino-6-benzyloxy-5-bromopyrimidine (3f)

2, 4-Diamino-5-bromo-6-chloropyrimidine (Phillips et. al., J. Org.Chem., 29, 1488-1490 (1963)) (2.3 g, 10 mmol) was added to a solution ofsodium (0.29 g, 12.5 mmol) in benzyl alcohol (10 mL) under argon. Thesolution was heated in a 130° C. oil bath for 3 h and the benzyl alcoholwas evaporated under reduced pressure to give a white solid. This solidwas washed with water, and air dried. Crystallization from 50% aqueousethanol gave white crystalline needles of 3f: yield, 2.32 g (76%); mp165-166° C. (lit. 136° C.; Kosary et. al., Acta Phazm. Hung., 49,241-247 (1989)); UV (pH 1) 1_(max) 236 nm (e=0.873×10⁴), 291(1.388×10⁴); (pH 6.9) 236 (0.850×10⁴), 277 (0.835×10⁴); (pH 13) 234(0.869×10⁴), 277 (0.827×10⁴); ¹H NMR d 5.30 (s, 2 H, ArCH₂), 6.15 (s, 2H, NH₂, exchange with D₂O), 6.32 (s, 2 H, NH₂, exchange with D₂O),7.31-7.45 (m, 5 H, ArH); MS (El) calcd. m/z for C₁₁H₁₁N₄O⁷⁹Br 294.0115,found 294.0127; calcd. m/z for C₁₁H₁₁N₄O⁸¹Br 296.0094, found 296.0083;Anal. (C₁₁H₁₁N₄OBr) C, H, N.

EXAMPLE 18

2-Amino-4-chloro-5-nitropyrimidine

A suspension of 2-amino-4-hydroxy-5-nitropyrimidine (5.0 g, 32.1 mmol)in phosphorous oxychloride (100 mL) was refluxed overnight, and theexcess phosphorous oxychloride was evaporated under reduced pressure.The residue was mixed with ice (100 g) in an ice-bath, and the mixturewas neutralized with concentrated aqueous sodium carbonate solution. Ayellow precipitate was collected by filtration and washed with water:yield, 1.39 g (25%); mp 191-194° C. dec.; ¹H NMR d 8.45 (br s, 2 H, NH₂,exchange with D₂O), 9.03 (s, 1 H, H-6); MS (EI) calcd. nmz for C₄H₃N₄O₂³⁵Cl 173.9944, found 173.9934; calcd. m/z for C₄H₃N₄O₂ ³⁷Cl 175.9915,found 175.9916.

EXAMPLE 19

2-Amino-4-benzyloxy-5-nitropyrimidine (5a)

2-Amino-4-chloro-S-nitropyrimldine (0.70 g, 4.0 mmol) was added to asolution of sodium (0.12 g, 5.2 mmol) in benzyl alcohol (8 mL) underargon. The solution was heated in a 130° C. oil bath for 3 h, andapproximately half of the benzyl alcohol was evaporated under reducedpressure. The residue was poured into water (50 mL) with constantstirring for 10 min. After neutralization with glacial acetic acid, abrown precipitate formed which was collected by filtration and washedwith water. This solid was crystallized from benzene to give 5a as agolden crystalline solid: yield, 126 mg (13%); mp 164-167° C.; UV (pH 1)1_(max) 262 nm (e=0.879×10⁴), 295 (sh) (0.571×10⁴); (pH 6.9) 235 (sh)(0.448×10⁴) 273 (0.360×10⁴), 326 (1.085×10⁴); (pH 13) 273 (0.404×10⁴),327 (1.055×10⁴); ¹H NMR d 5.51 (s, 2 H, ArCH₂), 7.35-7.54 (m, 5 H, ArH),8.05 (d, 2 H, NH₂, exchange with D₂O), 8.92 (s, 1 H, H-6); MS (EI)calcd. m/z for C₁₁H₁₀N₄O₃ 246.0752, found 246.0758; Anal. (C₁₁H₁₀N₄O₃)C, H, N.

EXAMPLE 20

2-Amino-4-benzyloxy-6-methyl-5-nitropyrimidine (5b)

2-Amino-4-chloro-6-methyl-5-nitropvrimidine (Boon et al., J. Chem. Soc.,96-102 (1951)) (1.24 g, 6.58 mmol) was added to a solution of sodium(0.21 g, 9.13 mmol) in benzyl alcohol (14 mL) under argon. The solutionwas heated in a 135° C. oil bath for 3.5 h, and was poured into water(70 mL) with constant stirring for 10 min. After neutralization withglacial acetic acid, a yellow precipitate formed which was collected byfiltration and washed with water. This solid was crystallized frombenzene to give 5b as a bright yellow crystalline solid: yield, 0.57 g(33%); mp 159-160° C.; UV (pH 1) 1_(max) 268 nm (e=0.783×10⁴), 345 (sh)(0.104×10⁴); (pH 6.9) 282 (0.564×10⁴), 345 (sh) (0.338×10⁴); (pH 13) 282(0.549×10⁴), 345 (sh) (0.332×10⁴); ¹H NMR d 2.35 (s, 3 H, CH₃), 5.44 (s,2 H, ArCH₂), 7.34-7.46 (m, 5 H, ArH), 7.64 (b s, 2 H, NH₂, exchange withD₂O); MS (EI) calcd. m/z for C₁₂H₁₂N₄O₃ 260.0908, found 260.0913; Anal.(C₁₂H₁₂N₄O₃) C, H, N.

EXAMPLE 21

2,4-Diamino-6-benzyloxy-s-triazine (6)

2,4-Diamino-6-chloro-s-triazine (2.25 g, 15.0 mmol) was added to asolution of sodium (0.43 g, 18.8 mmol) in benzyl alcohol (30 mL) underargon. The suspension was heated in a 130° C. oil bath for 3.5 h. Theexcess benzyl alcohol was removed under vacuum and the resulting solidwas collected with the aid of benzene, and washed with water (100 mL):yield, 1.83 g (56%); mp 184-185° C. (lit. 186-188° C.; Wakabayashi etal., Nippon Dojo-Hiryogaku Zasshi, 41, 193-200 (1970)); UV. (pH 1)1_(max) 233 nm (sh) (e=0.589×10⁴); (pH 6.9) 238 (sh) (0.111×10⁴)i (pH13) 240 (sh) (0.073×10⁴) ; ¹H NMR d 5.25 (s; 2 H, ArCH₂), 6.63 (s, 4 H,NH₂, exchange with D₂O), 7.30-7.42 (m, 5 H, ArH); MS (EI) calcd. m/z forC₁₀H₁₁N₅O 217.0963, found 217.0955.

EXAMPLE 22

2-Amino-6-chloro-8-trifluoromethylpurine

A suspension of 8-trifluoromethylguanine (Pfleiderer and Shanshal,Liebigs Ann. Chem., 726, 201-215 (1969)) (2.0 g, 9.1 mmol) inphosphorous oxychloride (20 mL) was refluxed for 3 h. Excess phosphorousoxychloride was evaporated under reduced pressure. The resulting residuewas mixed with ice-water (100 g), and the pH was adjusted to 3-4 with aconcentrated aqueous NaOH solution. The resulting solution was mixedwith MeOH (100 mL) and approximately half (i.e., 100 mL) or the aqueousmethanol solution was loaded on a 3×80 cm Sephadex LH-20 column elutedwith methanol/water (1:1) at 1 mL/min. Column eluent was continuouslymonitored at 280 nm and fractions (10 mL) were collected. The remainderof the reaction mixture in MeOH/H₂O was chromatographed separately underidentical conditions. The desired product eluted in fractions 73-85.Evaporation of solvent from the pooled fractions 73-85 from bothchromatographic nins afforded analytically pure2-amino-6-chloro-8-trifluoromethylpurine: yield, 0.94 g (43%); mp >225°C. dec.; UV (pH 1) 1_(max) 245 nm (e=0.501×10⁴), 314 (0.746×10⁴); (pH6.9) 270 (0.265×10⁴), 315 (0.612×10⁴); (pH 13) 272 (0.269×10⁴) 314(0.612×10⁴); 1H NMR d 7.19 (s, 2 H, NH₂, exchange with D₂O), 14.25 (brs, 1 H, NH, exchanges with D₂O); MS (EI) calcd. m/z for C₆H₃N₅F₃ ³⁷Cl237.0029, found 237.0011; calcd. m/z for C₆H₃N₅F₃ ³⁷Cl 239.0000, found238.9987; Anal. (C₆H₃N₅F₃C) C, H, N, F, Cl.

EXAMPLE 23

O⁶-Benzyl-8-trifluoromethylguanine (1e)

Sodium (0.10 g,. 4.3 mmol) was stirred in 5 mL of benzyl alcohol untilall had reacted. 2-Amino-6-chloro-8-trifluoromethylpurine (0.475 g, 2.0mmol) was added, and the reaction mixture was heated in a 135° C. oilbath for 3.5 h. The benzyl alcohol was removed by vacuum distillationyielding a brown oil. The oil was dissolved in water (50 mL) and wasacidified with glacial acetic acid to produce a pale yellow precipitate.The precipitate was collected by filtration and washed with water. Thecrude product was loaded on a 2.5×35 cm silica gel column (Davisil grade633, 200-425 mesh, 60 A). Elution was carried out with 5% EtOH in CHCI₃to provide analytically pure O⁶-benzyl-8-trifluoromethylguanine (1e):yield, 0.42 g (67%); mp 214-216° C. dec.; UV (pH 1) 1_(max) 291 nm(e=1.229×10⁴); (pH 6.9) 244 (0.470×10⁴), 289 (1.023×10⁴); (pH 13) 247(sh) (0.393×10⁴), 290 (0.923×10⁴); ¹H NMR d 5.51 (s, 2 H, ArG₂), 6.82(s, 2 H, NH₂, exchange with D₂O), 7.38-7.55 (m, 5 H, ArH), 13.75 (br s,1 H, NH, exchanges with D₂O); MS (EI) calcd. m/z for C₁₃H₁₀N₅OF₃309.0837, found 309.0827; Anal. (C₁₃H₁₀N₅OF₃) C, H, N, F.

EXAMPLE 24

O⁶-Benzyl-8-trifluoromethyl-9-methylguanine (7)

To O⁶-benzyl-8-trifluoromethylguanine (1e) (200 mg, 0.65 mmol) underargon was added 0.66 mL of a 1.0 M solution of sodium ethoxide inethanol. The solution was stirred for 10 min and the ethanol was removedunder vacuum. The remaining solid was dissolved in anhydrous DMF (1.5mL), and methyl iodide (49 μL, 0.78 mmol) was added to the solution.This solution was stirred at room temperature for 1 h, and 1.5 mLadditional DMF was added. The solution was stirred at room temperatureovernight. The solvent was evaporated under reduced pressure. The crudesolid was loaded on a 2.5×35 cm silica gel column (Davisil grade 633,200-425 mesh, 60 A). Elution was carried out with chloroform/hexane(3:1) to provide analytically pureO⁶-benzyl-8-trifluoromethyl-9-methylguanine (7): yield, 95 mg (45%); mp86-89° C.; UV (pH 1) 1_(max) 244 nm (e=0.581×10⁴), 286 (1.274×10⁴); (pH,6.9) 252 (0.608×10⁴), 288(1.022×10⁴); (pH 13) 252 (0.618×10⁴), 288(1.038×10⁴); ¹H NMR d 3.70 (s, 3 H, CH₃), 5.51 (s, 2 H, ArCH₂), 6.91 (s,2 H, NH₂, exchange with D₂O), 7.38-7.54 (m, 5 H, ArH); MS (El) calcd.m/z for C₁₄H₁₂N₃OF₃ 323.0994, found 323.0978; Anal. (C₁₄H₁₂N₃OF₃) C, H,N, F.

EXAMPLE 25

8-Aza-O⁶-benzyl-9-methylguanine (8a)

8-Aza-O⁶-benzylguanine (0.484 g, 2.0 mmol) was mixed. with 4 mL of 0.5 Msodium ethoxide in ethanol and stirred for 30 min. The ethanol wasevaporated under reduced pressure. The residue was dissolved inanhydrous DMF (6 mL), and methyl iodide (0.15 mL, 2.4 mmol) was added.The clear solution became cloudy within 10 min, and the resultingmixture was stirred overnight at room temperature. DMF was evaporatedunder reduced pressure to give a brown solid. The solid was dissolved inchloroform and loaded on a silica gel column (Davisil grade 633, 200-425mesh, 60 A). Product 8a was eluted with chloroform; yield, 138 mg (27%);mp 178-179° C.; UV (pH 1) 1_(max) 243 nm (sh) (e=0.556×10⁴), 284(1.112×10⁴); (pH 6.9) 243 (0.553×10⁴), 290 (0.998×10⁴); (pH 13) 242(0.549×10⁴), 290 (1.010×10⁴); ¹H NMR 3.96 (s, 3 H, CH₃), 5.57 (s, 2 H,ArCH₂), 7.18 (s, 2 H, NH₂, exchange with D₂O) 7.38-7.57 (m, 5 H, ArH);MS (EI) calcd m/z for C₁₂H₁₂N₆O 256.1072, found 256.1086; Anal.(C₁₂H₁₂N₆O) C, H, N.

EXAMPLE 26

8-Aza-O⁶-benzyl-9-(pivaloyloxymethyl) guanine (8b) and8-Aza-O⁶-benzyl-7-(pivaloyloxymethyl)guanine (9)

8-Aza-O⁶-benzylguanine (0.484 g, 2.0 mmol) was mixed with 4 mL of 0.5 Msodium ethoxide in ethanol and stirred for 30 min. The ethanol wasevaporated under reduced pressure. The residue was dissolved inanhydrous DMF (6 mL), and chloromethyl pivalate (0.3 mL, 2.1 mmol) wasadded. The clear solution was stirred for 8 h at room temperature. DMFwas evaporated under reduced pressure to give a brown solid. The solidwas dissolved in chloroform and loaded on a silica gel column (Davisilgrade 633, 200-425 mesh, 60 A). The 9-isomer (8b) was eluted from thecolumn with CHCl₃:hexane (4:1) while the 7-isomer (9) was subseuentlyeluted with CHCl₃. 8-Aza-O⁶-benzyl-9-(pivaloyloxymethyl)guanine (8b):yield, 405 mg (57%); mp 119-120° C.; UV (pH 1) 1_(max) 246 nm(e=0.494×10⁴), 286 (0.878×10⁴); (pH 6.9) 247 (0.472×10⁴), 288(0.819×10⁴); (pH 13) (decomposes to 8-aza-O⁶-benzylguanine); ¹H NMR d1.10 (s, 9 E, C(CH₃)₃), 5.50 (s, 2 H, ArCH₂), 6.31 (s, 2H, CH₂), 7.38(s, 2 H, NH₂, exchange with D₂O), 7.40-7.54 (m, 5 H, ArH); MS (EI) calcdm/z for C₁₇H₂₀N₆O₃ 356.1596, found 356.1578; Anal. (C₁₇H₂₀N₆O₃.{fraction (1/5+L )}H₂O) C, H, N.8-Aza-O⁶-benzyl-7-(pivaloyloxymethyl)guanine (9): yield, 103 mg (15%);mp 153-154° C.; UV (pH 1) 1_(max) 244 nm (e=0.820×10⁴), 294 (1.249×10⁴); (pH 6.9) 250 (sh) (0.296×10⁴) 313 (0.503×10⁴); (pH 13) (decomposes to8-aza-O⁶-benzylguanine); ¹H NMR d 1.12 (s, 9 H, C(CH₃)₃), 5.56 (s, 2 H,ArCH₂), 6.40 (s, 2 H, CH₂), 7.04 (s, 2 H, NH₂, exchange with D₂O),7.4-7.58 (m, 5 H, ArH); MS (EI) calcd m/z for C₁₇h₂₀ _(N) ₆O₃ 356.1596,found 356.1602; Anal. (C₁₇H₂₀N₆O₃).

EXAMPLE 27

O⁶-Benzyl-8-bromo-9-methylguanine (10a)

O⁶-Benzyl-9-methylguanine (0.252 g, 1.0 mmol) and sodium bicarbonate(0.084 g, 1.0 mmol) were dissolved in anhydrous DMF (2 mL) under argon.Bromine (52 mL, 1.0 mmol) was added to the solution and the resultingmixture was stirred overnight at room temperature. The solvent wasevaporated under reduced pressure. The residue was dissolved inchloroform, and loaded on a silica gel column (Davisil grade 633,200-425 mesh, 60 A). Product 10a was eluted with chloroform; yield, 180mg (52%); mp 150-152° C.; UV (pH1) 1_(max) 248 nm (e=0.753×10⁴), 292(1.398×10⁴); (pH 6.9) 251 (0.919×10⁴), 287 (1.306×10⁴); (pH 13) 251(0.906×10⁴), 287 (1.296×10⁴); ¹H NMR d 3.53 (s, 3 H, CH₃), 5.47 (s, 2 H,ArCH;), 6.61 (s, 2 H, NH₂, exchange with D₂O), 7.35-7.52 (m, 5 H, ArH);MS (EI) calcd m/z for C₁₃H₁₂N₅O⁷⁹Br 333.0225, found 333.0228; calcd m/zfor C₁₃H₁₂N₅O⁸¹ Br 335.0205, found 335.0188; Anal. (C₁₃H₁₂N₅OBr) C, H,N, Br:

EXAMPLE 28

O⁶-Benzyl-8-bromo-9-(pivaloyloxmethyl) guanine (10b) andO⁶-benzyl-8-bromo-7-(pivaloyloxymthyl) guanine (11)

O⁶-Benzyl-8-bromoguanine (Chae et al., J. Med. Chem., 38, 342-347(1995)) (0.48 a, 1.5 mmol) was mixed with 1.5 mL of a 1.0 M solution ofsodium ethoxide in ethanol and was stirred for 20 min. The ethanol wasremoved under reduced pressure and the solid residue was dissolved inDMF (5 mL). Chloromethylpivalate (0.24 mL, 1.65 mmol) was then added andthe solution was stirred overnight. The DMF was removed under reducedpressure. The residue was dissolved in chloroform and was loaded on asilica gel column (Davisil grade 633, 200-425 mesh, 60A) eluted withchloroform. The 9-isomer (10b) eluted earlier than the 7-isomer withchloroform and 10b was recovered in pure for under these conditions.O⁶-Benzyl-8-bromo-9-(pivaloyloxymethyl)guanine (1b): yield, 150 mg(23%); mp 217-218° C. UV (pH 1) 1_(max) 250 nm (e=0.944×10⁴), 291(1.166×10⁴); (pH 6.9) 266 (0.916×10⁴), 295 (0.916×10⁴); (pH 13)decomposes to O⁶-benzyl-8-bromoguanine; ¹H NMR d 1.13 (s, 9H, C(CH₃)₃),5.48 (s, 2H, ArCH₂), 5.93 (s, 2H, CH₂), 6.80 (s, 2H, NH₂, exchange withD₂O), 7.35-7.52 (m, 5H, ArH). MS (EI) calcd m/z for C₁₈H₂₀N₅O₃ ⁷⁹Br433.0750, found 433.0725; calcd m/z for C₁₈H₂₀N₅O₃ ⁸¹Br 435.0729, found435.0672; Anal. (C₁₈H₂₀N₅O₃Br) C, H, N, Br. The recovered 7-isomer (11)was rechromatographed on a silica gel column (Davisil grade 633, 200-425mesh, 60 A) which was eluted first with CHCl₃/hexane (1:1) followed byCHCl₃ to recover O⁶-benzyl-8-bromo-7-(pivaloyloxymethyl)guanine (11).

EXAMPLE 29

O⁶-Benzyl-7-(pivaloyloxmethyl) guanine (12)

O⁶-Benzylguanine (2.41 g, 10 mmol) was mixed with 10 mL of a 1.0 Msolution of sodium ethoxide in ethanol and was stirred for 30 min. Theethanol was evaporated under reduced pressure. The residue was dissolvedin anhydrous DMF (30 mL), and chloromethyl- pivalate (Aldrich) (1.5 mL,10.4 mmol) was added. The clear solution was stirred overnight at roomtemperature. DMF was evaporated under reduced pressure to give a palepeach-colored solid. The solid was dissolved in chloroform/ethanol (9:1)and loaded on a silica gel column (Davisil grade 633, 200-425 mesh, 60A). The column was eluted with chloroform/ethanol (9:1) to elute the9-isomer (Chae et al., J. Med. Chem., 37, 342-347 (1994)) followed bythe 7-isomer. The 7-isomer (12) was further purified by silica gelcolumn chromatography (Davisil grade 633, 200-425 mesh, 60 A) usingchloroform/ethanol (98:2) as eluent: yield, 36 mg (1%); mp 166-168° C.dec; UV (pH 1) 1. 240 nm (sh) (e=0.656×10⁴), 290 (1.164×10⁴) ; (pH 6.9)240 (sh) (0.635×10⁴) 293 (0.528×10⁴); (pH 13) decomposes toO⁶-benzylguanine; ¹H NMR d 0.98 (s, 9 H, C(CH₃)₃), 5.51 (s, 2 H, ArCH₂),6.07 (s, 2 H, CH₂), 6.32 (s, 2 H, NH₂, exchange with D₂O), 7.36-7.58 (m,5 H, ArH), 8.25 (s, 1 H, H-8); MS (EI) calcd m/z for C₁₈H₂₁N₅O₃355.1644, found 355.1626.

What is claimed is:
 1. A compound of the formula

wherein R is selected from the group consisting of C₁-C₄ alkyl, C₁-C₆alkylcarbonyloxy C₁-C₄ alkyl, C₁-C₄ alkyloxycarbonyl C₁-C₄ alkyl,carboxy C₁-C₄ alkyl, cyano C₁-C₄ alkyl, aminocarbonyl C₁-C₄ alkyl,hydroxy C₁-C₄ alkyl, and C₁-C₄ alkyloxy C₁-C₄ alkyl.
 2. The compound ofclaim 1, wherein R is selected from the group consisting of C₁-C₄ alkyland C₁-C₆ alkylcarbonyloxy C₁-C₄ alkyl.
 3. The compound of claim 1wherein said compound is selected from the group consisting of8-aza-O⁶-benzyl-9-methylguanine and8-aza-O⁶-benzyl-9-(pivaloyloxymethyl) guanine.
 4. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of claim
 1. 5. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of claim
 2. 6. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of claim
 3. 7. A pharmaceutical composition ofclaim 4, wherein said pharmaceutically acceptable carrier comprisespolyethylene glycol.
 8. A method of enhancing the chemotherapeutictreatment of tumor cells in a mammal with antineoplastic alkylatingagent that causes cytotoxic lesions at the O⁶-position of guanine, whichmethod comprises: administering to a mammal an effective amount of acompound of claim 1, and administering to said mammal an effectiveamount of an antineoplastic alkylating agent which causes cytotoxiclesions at the O⁶-position of guanine.
 9. A method of enhancing thechemotherapeutic treatment of tumor cells in a mammal withantineoplastic alkylating agent that causes cytotoxic lesions at theO⁶-position of guanine, which method comprises: administering to amammal an effective amount of a compound of claim 2, and administeringto said mammal an effective amount of an antineoplastic alkylating agentwhich causes cytotoxic lesions at the O⁶-position of guanine.
 10. Amethod of enhancing the chemotherapeutic treatment of tumor cells in amammal with antineoplastic alkylating agent that causes cytotoxiclesions at the O⁶-position of guanine, which method comprises:administering to a mammal an effective amount of a compound of claim 3,and administering to said mammal an effective amount of anantineoplastic alkylating agent which causes cytotoxic lesions at theO⁶-position of guanine.
 11. A method of inhibiting the reaction ofO⁶-alkylguanine-DNA-alkyltransferase with an alkylated DNA comprisingreacting the O⁶-alkylguanine-DNA-alkyltransferase with the compound ofclaim
 1. 12. A compound of the formula

wherein said R is selected from the group consisting of C₁-C₄ alkyl,C₁-C₆ alkylcarbonyloxy C₁-C₄ alkyl, C₁-C₄ alkyloxycarbonyl C₁-C₄ alkyl,carboxy C₁-C₄ alkyl, cyano C₁-C₄ alkyl, aminocarbonyl C₁-C₄ alkyl,hydroxy C₁-C₄ alkyl, and C₁-C₄ alkyloxy C₁-C₄ alkyl.
 13. The compound ofclaim 12, wherein said R is C₁-C₆ alkylcarbonyloxy C₁-C₄ alkyl.
 14. Thecompound of claim 13, wherein said compound is8-aza-O⁶-benzyl-7-(pivaloyloxymethyl) guanine.
 15. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of claim
 12. 16. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of claim
 13. 17. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of claim
 14. 18. A pharmaceutical composition ofclaim 15, wherein said pharmaceutically acceptable carrier comprisespolyethylene glycol.
 19. A method of enhancing the chemotherapeutictreatment of tumor cells in a mammal with antineoplastic alkylatingagent that causes cytotoxic lesions at the O⁶-position of guanine, whichmethod comprises: administering to a mammal an effective amount of acompound of claim 12, and administering to said mammal an effectiveamount of an antineoplastic alkylating agent which causes cytotoxiclesions at the O⁶-position of guanine.
 20. A method of enhancing thechemotherapeutic treatment of tumor cells in a mammal withantineoplastic alkylating agent that causes cytotoxic lesions at theO⁶-position of guanine, which method comprises: administering to amammal an effective amount of a compound of claim 13, and administeringto said mammal an effective amount of an antineoplastic alkylating agentwhich causes cytotoxic lesions at the O⁶-position of guanine.
 21. Amethod of enhancing the chemotherapeutic treatment of tumor cells in amammal with antineoplastic alkylating agent that causes cytotoxiclesions at the O⁶-position of guanine, which method comprises:administering to a mammal an effective amount of a compound of claim 14,and administering to said mammal an effective amount of anantineoplastic alkylating agent which causes cytotoxic lesions at theO⁶-position of guanine.
 22. A method of inhibiting the reaction ofO⁶-alkylguanine-DNA-alkyltransferase with an alkylated DNA comprisingreacting the O⁶-alkylguanine-DNA-alkyltransferase with the compound ofclaim 1.